Compositions for treatment of vascular disease

ABSTRACT

Provided are various embodiments relating to compositions and methods for treating vascular disease, including core NOX1 promoters and variants thereof for regulating expression of transgenes in response to vascular pathology and allowing for increased transgene loading capacity. Also provided are variant FOXP polypeptides having a zinc finger and leucine zipper region of a different FOXP polypeptide. Further provided are vectors comprising the core NOX1 promoters and/or a coding sequence for variant FOXP polypeptides described herein and optionally coding sequence(s) for one or more additional therapeutic polypeptide(s), such as IL10, for treating inflammation-associated diseases, such as vascular disease. Also provided is a screening model for testing therapeutic agents capable of treating established and ongoing atherosclerotic pathology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/078,163, filed Oct. 23, 2020, which claims the benefit of priority ofU.S. Provisional Application No. 63/013,869, filed Apr. 22, 2020, eachof which is incorporated by reference herein in its entirety for anypurpose.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled2020-10-19_01258-0001-00US-T1_ST25.txt created Oct. 19, 2020, which is51,001 bytes in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

FIELD

The present disclosure relates to compositions and methods for treatingvascular disease, and potentially other inflammation-associated diseasessuch as asthma, diabetes, arthritis, dementia, Alzheimer's disease,macular degeneration, age-related diseases, etc. The present disclosurealso relates to promoters for regulating expression of transgenes inresponse to vascular pathology, including core NOX1 promoters andvariants thereof described herein. The present disclosure furtherrelates to vectors and expression systems comprising a core NOX1promoter described herein, including, for example, AAV vectors havingincreased loading capacity for expressing multiple proteins. The presentdisclosure further relates to variant FOXP polypeptides having a zincfinger and leucine zipper region of a different FOXP polypeptide. Inaddition, the present disclosure relates to nucleic acids, vectors, andexpression systems encoding FOXP polypeptides described herein andmethods of using the same (e.g., gene therapy methods), for exampleregulated expression of FOXP polypeptides. The present disclosure alsorelates to a treatment model for establishing atherosclerotic pathologyprior to administering a therapeutic agent to identify those capable oftreating established as well as preventing ongoing atheroscleroticpathology.

BACKGROUND

Some disorders, such as cystic fibrosis and sickle cell disease, arecaused by mutations in a single gene. Multifactorial disorders, on theother hand, are not caused by a single genetic cause and are much morecomplex. Multifactorial disorders or conditions, such as heart disease,inflammation, cancer, dementia, obesity, and type 2 diabetes, are likelyinfluenced by multiple genes (polygenic) in combination with lifestyleand environmental factors.

The field of gene therapy has experienced recent successes in thetreatment of single-gene disorders. Gene therapy generally involvesusing a carrier (e.g., a virus) to deliver a therapeutic gene (or genes)to target cells for expression of the encoded therapeutic agent(s) forthe treatment and/or prevention of a disease or condition.Adeno-associated virus (AAV) is a preferred gene therapy vector becauseof its proven gene delivery effect, low immunogenicity, and apparentlack of pathogenicity. The generation and use of recombinant AAV tocarry and deliver foreign genes into cells, animals, and humans was madepossible by foundation work involving AAV molecular biology (Batchu R B,et al., 1995; Cao M, et al., 2014; Hermonat P L, et al., 1984a, 1984b,1996, 1997a, 1997b, 1997c, 1998, 2014; Labow M A, et al., 1986; LaFaceD, et al., 1988), and the first AAV-based gene delivery conducted in1984 (Hermonat P L, et al., 1984b, 2014). However, AAV's limited 4.7 kbpackaging capacity restricts the size and number of promoter elementsand coding sequences that can be incorporated into a single vector.Hermonat P L, et al., 1997c. For instance, the inverted terminal repeatsand support sequences, such as multiple cloning sites and poly Asequences, alone can take up about 500-600 bp. Hence, there is a need todevelop AAV vectors that can deliver multiple genes for the treatmentand/or prevention of multifactorial disorders.

AAV is capable of infecting a large range of host cells (both dividingand quiescent) and persisting in an extrachromosomal state withoutintegrating into the genome of the host cells. As a result, AAV deliveryof highly active therapeutic genes under the control of a constitutivepromoter (e.g., a CMV promoter) can cause unwanted expression of atherapeutic protein in non-target cells. As a result, regulatedpromoters that respond to specific stimuli are preferred to control thein vivo expression profile of therapeutic genes. Hence, there is a needto develop small regulated promoters that are responsive to stimulicharacteristic of the disease or condition being treated, and/or thatare responsive to stimuli characteristic of a particular target celltype.

High cholesterol (characteristic of high lipid diets) has beenassociated with increased risk of cardiovascular disease (includingcoronary heart disease, stroke, and peripheral vascular disease),diabetes, and high blood pressure. Other multifactorial disorders, suchas obesity, fatty liver disease, cancer, and age-related memory loss,may also be associated with high-lipid diets. The additive effects of ahigh-lipid diet, including elevated levels of reactive oxygen species(ROS), also referred to as oxidative stress, may accumulate slowly overtime resulting in delayed diagnosis of the high-lipid associateddisorder(s). Gregersen S, et al., 2012. For example, atherosclerosis, isa chronic inflammatory disease of the blood vessels involving thegradual buildup of fatty material on the inner wall of arteries andinfiltration of lymphocytes and macrophages causing restricted bloodflow. Macrophages penetrate past the endothelial cells of the bloodvessels, localize on blood vessel walls, take up (or internalize)oxidized-low density lipoprotein (Ox-LDL) and other lipids throughscavenger receptors, and retain the lipid in vesicles. These lipid-ladencells or “foam cells” are a major component of plaque. Unfortunately,high-lipid associated vascular pathologies (such as atherosclerosis) maygo undiagnosed until the subject suffers a stroke or heart attack.

There is a need to develop an appropriate animal model of high-lipidassociated disorders to test in vivo which therapies may be capable oftreating, reducing, and/or reversing the additive and long-term effectsresulting from high-lipid diets. For example, prior animal studies usingthe low density lipoprotein receptor knockout mouse (LDLR-KO) involveintroducing the gene therapy vector on or near the same day that theanimals are placed on a high cholesterol diet (HCD). Cao M, et al.,2015; Zhu H, et al., JTM 2014; Zhu H, et al., Plos One 2014; Cao M, etal., 2011; Khan J A, et al., 2011; Khan J A, et al., 2010; Dandapat A,et al., BBRC, 2007; Dandapat A, et al., Gene Ther, 2007; Liu Y, et al.,2005. However, because it takes time for a HCD to induce atheroscleroticpathology, the prior model of administering the vector and starting theHCD at the same time cannot test whether the vector is capable oftreating, reducing, and/or reversing significant and establishedatherosclerotic pathology. A treatment model more realistic of thelong-term clinical pathology observed with high-lipid associateddisorders is needed for developing effective therapies.

Exemplary promoters, therapeutic proteins, vectors (including multi-genevectors), therapeutic methods, and clinically-relevant animal models fortreatment and prevention of high-lipid associated disorders aredisclosed herein.

Unlike prior models, the treatment models described herein involveestablishing atherosclerotic pathology prior to administering atherapeutic agent (such as a gene therapy vector) to identifytherapeutic agents capable of treating, reducing, and/or reversingestablished as well as preventing ongoing atherosclerotic pathology.

Also described herein are AAV vectors capable of expressing multipletherapeutic genes, such as anti-inflammatory genes, in the presence of ahigh-lipid environment. For example, core NOX1 promoters were identifiedand developed to include additional transcriptional elements whilemaintaining a size small enough for AAV packaging capacity of multiplecoding sequences. As described herein, an exemplary core NOX1 promoterwas shown to regulate expression of two anti-inflammatory proteins, anexemplary chimeric human FOXP3/FOXP1 polypeptide and human IL10, in thepresence of a high-lipid environment. The therapeutic construct waspackaged into an AAV vector and tested in the animal model ofestablished atherosclerotic pathology described herein. High resolutionultrasound measurement of systolic blood velocity, lumen cross sectionalarea, and wall thickness coupled with histological analysis showedsignificantly less vascular pathology in the AAV-FOXP3(P1)-IL10-treatedmice compared to the control vector-treated mice.

SUMMARY

Embodiment 1. An isolated nucleic acid molecule comprising a NOX1 corepromoter and a heterologous nucleic acid molecule, wherein the NOX1 corepromoter comprises a nucleotide sequence having at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

Embodiment 2. The isolated nucleic acid molecule of embodiment 1,wherein the NOX1 core promoter is less than 600 nucleotides, less than550 nucleotides, less than 500 nucleotides, less than 480 nucleotides,or less than 470 nucleotides.

Embodiment 3. The isolated nucleic acid molecule of any one of thepreceding embodiments, comprising at least one heterologous NFκB bindingsite.

Embodiment 4. The isolated nucleic acid molecule of embodiment 3,wherein the at least one heterologous NFκB binding site comprises thenucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20.

Embodiment 5. The isolated nucleic acid molecule of embodiment 3 orembodiment 4, wherein the at least one NFκB binding site comprises thenucleotide sequence of SEQ ID NO: 5.

Embodiment 6. The isolated nucleic acid molecule of any one of thepreceding embodiments, comprising at least two, at least three, at leastfour, or at least five heterologous NFκB binding sites.

Embodiment 7. The isolated nucleic acid molecule of any one of thepreceding embodiments, comprising at least one heterologous Oct1 bindingsite.

Embodiment 8. The isolated nucleic acid molecule of embodiment 7,wherein the at least one Oct1 binding site comprises the nucleotidesequence of SEQ ID NO: 21.

Embodiment 9. The isolated nucleic acid molecule of any one of thepreceding embodiments, comprising at least two, at least three, at leastfour, or at least five Oct1 binding sites.

Embodiment 10. An isolated nucleic acid molecule comprising a nucleotidesequence having at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to thenucleotide sequence of SEQ ID NO: 4.

Embodiment 11. The isolated nucleic acid molecule of any one of thepreceding embodiments, comprising the nucleotide sequence of SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO: 4.

Embodiment 12. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 2 and a heterologous nucleotidesequence.

Embodiment 13. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 3.

Embodiment 14. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 4.

Embodiment 15. The isolated nucleic acid molecule of embodiment 13 orembodiment 14, comprising a heterologous nucleic acid molecule.

Embodiment 16. The isolated nucleic acid molecule of any one of thepreceding embodiments, wherein the heterologous nucleic acid moleculecomprises a heterologous nucleotide sequence encoding ananti-inflammatory molecule or reporter protein.

Embodiment 17. The isolated nucleic acid molecule of embodiment 16,wherein the anti-inflammatory molecule is a wildtype or variant FOXP3polypeptide and/or IL10.

Embodiment 18. The isolated nucleic acid molecule of embodiment 17,wherein the wildtype or variant FOXP3 polypeptide comprises the aminoacid sequence of SEQ ID NO: 24 or SEQ ID NO: 28.

Embodiment 19. An isolated variant FOXP polypeptide comprising the aminoacid sequence of a first FOXP polypeptide, wherein a zinc finger andleucine zipper region of the first FOXP polypeptide has been replacedwith a zinc finger and leucine zipper region of a second FOXPpolypeptide.

Embodiment 20. The isolated variant FOXP polypeptide of embodiment 19,wherein:

a) the first FOXP polypeptide is a FOXP3 polypeptide and the second FOXPpolypeptide is a FOXP1 polypeptide, a FOXP2 polypeptide, or a FOXP4polypeptide;

b) the first FOXP polypeptide is a FOXP1 polypeptide and the second FOXPpolypeptide is a FOXP2 polypeptide, a FOXP3 polypeptide, or a FOXP4polypeptide;

c) the first FOXP polypeptide is a FOXP2 polypeptide and the second FOXPpolypeptide is a FOXP1 polypeptide, a FOXP3 polypeptide, or a FOXP4polypeptide; or

-   -   d) the first FOXP polypeptide is a FOX4 polypeptide and the        second FOXP polypeptide is a FOXP1 polypeptide, a FOXP2        polypeptide, or a FOXP3 polypeptide.

Embodiment 21. The isolated variant FOXP polypeptide of embodiment 19 orembodiment 20, wherein the first FOXP polypeptide is a FOXP3 polypeptideand the second FOXP polypeptide is a FOXP1 polypeptide.

Embodiment 22. The isolated variant FOXP polypeptide of any one ofembodiments 19 to 21, wherein the zinc finger and leucine zipper regionof the second FOXP polypeptide comprises the amino acid sequence of SEQID NO: 27.

Embodiment 23. The isolated variant FOXP polypeptide of any one ofembodiments 19 to 22, comprising an amino acid sequence having at least97%, at least 98%, at least 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 28.

Embodiment 24. The isolated variant FOXP polypeptide of any one ofembodiments 19 to 23, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO: 28.

Embodiment 25. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 28.

Embodiment 26. An isolated nucleic acid comprising the nucleotidesequence of SEQ ID NO: 29 or SEQ ID NO: 34.

Embodiment 27. An isolated nucleic acid comprising a nucleic acidsequence encoding the variant FOXP polypeptide of any one of embodiments19 to 25.

Embodiment 28. An isolated contiguous nucleic acid comprising a firstnucleic acid sequence encoding a first therapeutic polypeptide and asecond nucleic acid sequence encoding a second therapeutic polypeptide,wherein the first therapeutic polypeptide comprises the variant FOXPpolypeptide of any one of embodiments 19 to 25.

Embodiment 29. The isolated nucleic acid of embodiment 28, wherein thesecond nucleic acid sequence is downstream of the first nucleic acidsequence.

Embodiment 30. The isolated nucleotide of embodiment 28, wherein thefirst nucleic acid sequence is downstream of the second nucleic acidsequence.

Embodiment 31. The isolated nucleic acid of any one of embodiments 28 to30, wherein the second therapeutic polypeptide is a signaling protein,such as a cytokine, a growth factor, or a chemokine.

Embodiment 32. The isolated nucleic acid of any one of embodiments 28 to31, wherein the second therapeutic polypeptide is an IL10 polypeptide.

Embodiment 33. The isolated nucleic acid of any one of embodiments 28 to32, wherein the second therapeutic polypeptide comprises the amino acidsequence of SEQ ID NO: 31.

Embodiment 34. The isolated nucleic acid of any one of embodiments 28 to33, wherein the first nucleic acid sequence and/or the second nucleicacid sequence is operatively linked to a first promoter.

Embodiment 35. The isolated nucleic acid of any one of embodiments 28 to34, wherein the second nucleic acid sequence is operatively linked to asecond promoter.

Embodiment 36. The isolated nucleic acid of embodiment 35 or embodiment35, wherein the first promoter and/or the second promoter is aconstitutive promoter.

Embodiment 37. The isolated nucleic acid of any one of embodiments 34 to36, wherein the first promoter and/or the second promoter is acytomegalovirus (CMV) immediate early promoter, a simian virus 40 (SV40)early promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a humanβ-actin promoter, or a chicken β-actin promoter and CMV early enhancer.

Embodiment 38. The isolated nucleic acid of any one of embodiments 34 to37, wherein the first promoter and/or the second promoter is a regulatedpromoter.

Embodiment 39. The isolated nucleic acid of any one of embodiments 34 to38, wherein the first promoter and/or the second promoter is atissue-specific promoter or a pathology-specific promoter.

Embodiment 40. The isolated nucleic acid of any one of embodiments 34 to39, wherein the first promoter and/or the second promoter is activatedby sheer stress and/or dyslipidemia.

Embodiment 41. The isolated nucleic acid of any one of embodiments 34 to40, wherein the first promoter and/or the second promoter is activatedby Angiotensin-2 (Ang II), Lipopolysaccharides (LPS), Oxidized LowDensity Lipoprotein (Ox-LDL), and/or carbamylated LDL.

Embodiment 42. The isolated nucleic acid of any one of embodiments 34 to41, wherein the first promoter and/or the second promoter comprises thenucleic acid of any one of embodiments 1 to 15.

Embodiment 43. The isolated nucleic acid of any one of embodiments 26 to42, comprising a TATA box between the first nucleic acid sequence andthe second nucleic acid sequence.

Embodiment 44. The isolated nucleic acid of any one of embodiments 26 to43, comprising at least one polyadenylation sequence.

Embodiment 45. The isolated nucleic acid of any one of embodiments 26 to44, comprising adeno-associated virus (AAV) inverted terminal repeats.

Embodiment 46. A vector comprising the nucleic acid of any one ofembodiments 1 to 18 or any one of embodiments 26 to 45.

Embodiment 47. The vector of embodiment 46, wherein the vector is avirus vector. Embodiment 48. The vector of embodiment 46 or embodiment47, wherein the vector is an adeno-associated virus (AAV) vector, anadenovirus vector, a retrovirus vector, a herpesvirus vector, or a poxvirus vector.

Embodiment 49. The vector of any one of embodiments 46 to 48, whereinthe vector is an adeno-associated virus (AAV) vector.

Embodiment 50. The vector of any one of embodiments 46 to 49, whereinthe vector is an adeno-associated virus (AAV) vector having a capsidserotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and anyvariant thereof.

Embodiment 51. A cultured host cell comprising the nucleic acid moleculeof any one of embodiments 1 to 18 or any one of embodiments 26 to 45,the variant FOXP polypeptide of any one of embodiments 19 to 24, or thevector of any one of embodiments 46 to 50.

Embodiment 52. A pharmaceutical composition comprising the nucleic acidmolecule of any one of embodiments 1 to 18 or any one of embodiments 26to 45, the variant FOXP polypeptide of any one of embodiments 19 to 24,the vector of any one of embodiments 46 to 50, or the cultured host cellof embodiment 51.

Embodiment 53. A method comprising administering to a subject thenucleic acid molecule of any one of embodiments 1 to 18 or any one ofembodiments 26 to 45, the variant FOXP polypeptide of any one ofembodiments 19 to 24, the vector of any one of embodiments 46 to 50, thecultured host cell of embodiment 51, or the pharmaceutical compositionof embodiment 52.

Embodiment 54. A method of treating a subject comprising administeringto the subject the nucleic acid molecule of any one of embodiments 1 to18 or any one of embodiments 26 to 45, the variant FOXP polypeptide ofany one of embodiments 19 to 24, the vector of any one of embodiments 46to 50, the cultured host cell of embodiment 51, or the pharmaceuticalcomposition of embodiment 52.

Embodiment 55. The method of embodiment 53 or embodiment 54, wherein thesubject has a vascular disease and/or a cardiovascular disease.

Embodiment 56. The method of any one of embodiments 53 to 55, whereinthe subject has an inflammation-associated disease.

Embodiment 57. The method of any one of embodiments 53 to 56, whereinthe subject has an age-associated disease.

Embodiment 58. The method of any one of embodiments 53 to 57, whereinthe subject has atherosclerosis.

Embodiment 59. The method of any one of embodiments 53 to 58, whereinthe subject has arthritis, such as psoriatic arthritis, rheumatoidarthritis, and/or gouty arthritis.

Embodiment 60. The method of any one of embodiments 53 to 59, whereinthe subject has dementia.

Embodiment 61. The method of any one of embodiments 53 to 60, whereinthe subject has Alzheimer's disease.

Embodiment 62. The method of any one of embodiments 53 to 61, whereinthe subject has asthma.

Embodiment 63. The method of any one of embodiments 53 to 62, whereinthe subject has macular degeneration of the retina.

Embodiment 64. The method of any one of embodiments 53 to 63, whereinthe subject has arterial disease of the aorta, carotid artery disease,coronary artery disease, atherosclerotic cerebrovascular disease,peripheral artery disease, and/or diabetes mellitus.

Embodiment 65. A method of screening a therapeutic agent comprising:

a) maintaining a low density lipoprotein receptor knockout (LDLR-KO) orApo E knockout animal (such as a mouse or rat) on a high cholesteroldiet; and

b) administering a therapeutic agent to the animal no earlier than 56days after beginning the high cholesterol diet.

Embodiment 66. The testing method of embodiment 65, wherein the methodfurther comprises:

c) assessing the blood flow velocity, cross-sectional area of the aortalumen, and/or the aortic wall thickness of the animal by ultrasoundimaging.

Embodiment 67. The testing method of embodiment 65 or embodiment 66,wherein the therapeutic agent is administered no earlier than 63 days,no earlier than 70 days, no earlier than 77 days, no earlier than 84days, no earlier than 91 days, or no earlier than 98 days afterbeginning the high cholesterol diet.

Embodiment 68. The testing method of any one of embodiments 65 to 67,wherein the therapeutic agent is the nucleic acid of any one ofembodiments 1 to 18 or any one of embodiments 26 to 45, the variant FOXPpolypeptide of any one of embodiments 19 to 24, the vector of any one ofembodiments 46 to 50, or the pharmaceutical composition of embodiment51.

Embodiment 69. The method of any one of embodiments 53 to 68, whereinthe nucleic acid, the variant FOXP polypeptide, the vector, the culturedhost cell, the pharmaceutical composition, or the therapeutic agent isadministered to the subject or the animal once.

Embodiment 70. The method of any one of embodiments 53 to 69, whereinthe nucleic acid, the variant FOXP polypeptide, the vector, the culturedhost cell, the pharmaceutical composition, or the therapeutic agent isadministered to the subject or the animal every other day, weekly, ormonthly.

Embodiment 71. The method of any one of embodiments 53 to 70, whereinthe nucleic acid, the variant FOXP polypeptide, the vector, the culturedhost cell, the pharmaceutical composition, or the therapeutic agent isadministered to the subject or the animal via intravenous injection,arterial injection, intramuscular injection, or injection into a sectionof ligated artery or vein.

Embodiment 72. The method of any one of embodiments 53 to 71, wherein atherapeutically effective amount of the nucleic acid, the variant FOXPpolypeptide, the vector, the cultured host cell, the pharmaceuticalcomposition, or the therapeutic agent is administered to the subject orthe animal.

Embodiment 73. The method of any one of embodiments 53 to 72, whereinthe vector is administered to the subject or the animal at a dose of1×10¹⁰ encapsidated genomes, 1×10¹¹ encapsidated genomes, 1×10¹²encapsidated genomes, 1×10¹³ encapsidated genomes, 1×10¹⁰ to 1×10¹³encapsidated genomes, 1×10¹⁰ to 1×10¹² encapsidated genomes, 1×10¹¹ to1×10¹² encapsidated genomes, or 1×10¹⁰ to 1×10¹¹ encapsidated genomes.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice. The objects and advantageswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) andtogether with the description, serve to explain the principles describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides expression levels of FoxP3 mRNA by Quantitative RT-PCR(Q-RT-PCR) in immortal, tissue culture A7R5 rat smooth muscle cellstransfected with pSV40-Neo and CMV-FoxP3 expression plasmids andselected with G418.

FIG. 1B shows expression levels of TGFβ by Q-RT-PCR in A7R5 rat smoothmuscle cells expressing FOXP3.

FIG. 1C shows expression levels of IL10 by Q-RT-PCR in A7R5 rat smoothmuscle cells expressing FOXP3.

FIG. 2A shows relative expression levels of FLAG under the control ofthe eNOX1 promoter in the presence of increasing concentrations ofAngiotensin-2 (Ang II) compared to an untransfected control.

FIG. 2B shows relative expression levels of FLAG under the control ofthe eNOX1 promoter in the presence of increasing concentrations ofLipopolysaccharides (LPS) compared to an untransfected control.

FIG. 2C shows relative expression levels of FLAG under the control ofthe eNOX1 promoter in the presence of increasing concentrations ofOxidized Low Density Lipoprotein (Ox-LDL) compared to an untransfectedcontrol.

FIG. 3A shows expression of FLAG-mCherry in HEK 293 cells under thecontrol of the CMV promoter and an untransfected control.

FIG. 3B shows relative expression of FLAG-mCherry in HEK 293 cellstransfected with NOXpr-3xFLAG-mCherry and treated with increasingconcentrations of Ang II.

FIG. 3C shows relative expression of FLAG-mCherry in HEK 293 cellstransfected with NOXpr-3xFLAG-mCherry and treated with increasingconcentrations of LPS.

FIG. 3D shows relative expression of FLAG-mCherry in HEK 293 cellstransfected with NOXpr-3xFLAG-mCherry and treated with increasingconcentrations of ox-LDL.

FIG. 4A shows the design of an in vivo expression experiment in the lowdensity lipoprotein receptor knockout (LDLR-KO) mouse, as described inExample 3. Three groups of LDLR-KO mice were used in the 20-weekexperiment. Group 1 (n=12) was a negative control group of untreatedLDLR-KO mice maintained on a normal diet (ND) from day 0 to week 6(n=5), week 12 (n=4), or week 20 (n=3). Groups 2 and 3 receivedAAV2/8.eNOXpr-3xFLAG-mCherry on day 0, followed by two boosterinjections at an interval of approximately 2 days. Group 2 (n=14) was asecond negative control group and received the ND from day 0 to week 6(n=4), week 12 (n=4), or week 20 (n=6). Group 3 (n=15) received a highcholesterol diet (HCD) from day 0 to week 6 (n=4), week 12 (n=4), orweek 20 (n=7).

FIG. 4B shows the design of an in vivo therapeutic effect experiment inthe LDL-KO mouse, as described in Example 6. Three groups of LDLR-KOmice were used in a 20-week study. Group 1 was a negative control groupof untreated LDLR-KO mice maintained on a normal diet (ND) from day 0 toweek 20 (n=3). Groups 2 and 3 each received the HCD diet from day 0through week 20. At week 12, Group 2 received theAAV2/8.eNOXpr-3xFLAG-mCherry vector (disease positive control) (n=9) andGroup 3 received the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector (experimentalgroup) (n=9), followed by two booster injections at an interval ofapproximately 2 days.

FIG. 5A is a schematic illustrating an increase of disease progressionand gene expression over time in LDLR-KO mice fed a high cholesteroldiet and following injection with eNOX1 promoter vector.

FIG. 5B shows immunofluorescence staining for FLAG in liver of HCD-fedand ND-fed LDLR-KO mice following injection ofAAV2/8.eNOXpr-3xFLAG-mCherry at 6 weeks, 12 weeks, and 20 weekspost-injection (20× and 60× magnifications). The tissue wascounterstained with DAPI.

FIG. 5C shows a Western blot for FLAG in liver tissue of HCD-fed andND-fed LDLR-KO mice following injection of AAV2/8.eNOXpr-3xFLAG-mCherryat 20 weeks post-injection. Detection of mouse β-actin served as aloading control.

FIG. 5D shows a Western blot for FLAG in liver tissue of HCD-fed andND-fed LDLR-KO mice following injection of AAV2/8.eNOXpr-3xFLAG-mCherry.Results are shown for ND-fed mice at 6 weeks post-injection and forHCD-fed mice at 6, 12, and 20 weeks post-injection. Detection of mouseβ-actin served as a loading control.

FIG. 5E shows results of densitometric analysis of the Western blotanalysis shown in FIG. 5C. Data are expressed as mean±SD (*P<0.05).

FIG. 5F shows results of densitometric analysis of the Western blotanalysis shown in FIG. 5D. Data are expressed as mean±SD (*P<0.05).

FIG. 6 is a schematic illustrating eNOX1pr-FoxP3(P1)-IL10 (SEQ ID NO:34), a three-component expression construct totaling 2675 nucleotidesand comprising the eNOX1 promoter (SEQ ID NO: 4), FOXP3(P1) codingsequence (SEQ ID NO: 29), and a human IL10 coding sequence (SEQ ID NO:32).

FIG. 7 is a schematic illustrating an animal treatment model of existingand ongoing vascular pathology involving first establishing diseaseprogression by maintaining a LDLR-KO mouse on a HCD and thensubsequently administering a therapeutic vector (e.g.,AAV.eNOXpr-FoxP3(P1)-IL10) some weeks to months later. High resolutionultrasound may be used to monitor disease progression and treatment.

FIG. 8A and FIG. 8B show qRT-PCR analysis of FoxP3(P1) mRNA in livertissue (FIG. 8A) and heart tissue (FIG. 8B) of LDLR-KO mice that weremaintained on a HCD beginning at day 1, administeredAAV2/8.eNOXpr-FOXP3(P1)-IL10 or AAV2/8.eNOXpr-3xFLAG-mCherry at week 12,and harvested at week 20. FoxP3(P1) expression was normalized toexpression of GAPDH.

FIG. 8C and FIG. 8D show qRT-PCR analysis of IL10 mRNA in liver tissue(FIG. 8C) and heart tissue (FIG. 8D) of LDLR-KO mice that weremaintained on a HCD beginning at day 1, administeredAAV2/8.eNOXpr-FOXP3(P1)-IL10 or AAV2/8.eNOXpr-3xFLAG-mCherry at week 12,and harvested at week 20. FoxP3(P1) expression was normalized toexpression of GAPDH.

FIG. 9A shows concentration of total plasma cholesterol at 20 weeks forLDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 9B shows concentration of triglycerides at 20 weeks for LDLR-KOmice fed a normal diet or fed a high cholesterol diet and administeredat 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vector or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 9C shows concentration of low-density lipoprotein at 20 weeks forLDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 9D shows concentration of high-density lipoprotein at 20 weeks forLDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 10A shows weight at 20 weeks among LDLR-KO mice fed a normal dietor fed a high cholesterol diet and administered at 12 weeks either theAAV2/8.eNOXpr-3xFLAG-mCherry vector or the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector.

FIG. 10B shows levels of alanine aminotransferase at 20 weeks amongLDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 10C shows levels of aspartate aminotransferase at 20 weeks amongLDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 10D shows levels of alkaline phosphatase at 20 weeks among LDLR-KOmice fed a normal diet or fed a high cholesterol diet and administeredat 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vector or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 10E shows levels of albumin at 20 weeks among LDLR-KO mice fed anormal diet or fed a high cholesterol diet and administered at 12 weekseither the AAV2/8.eNOXpr-3xFLAG-mCherry vector or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 11A shows images of representative aortas stained with Oil Red Ofrom LDLR-KO mice fed a normal diet or fed a high cholesterol diet andadministered at 12 weeks either the AAV2/8.eNOXpr-3xFLAG-mCherry vectoror the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector.

FIG. 11B shows enlarged images of the aortic arch region of the Oil RedO-stained aortas shown in FIG. 11A.

FIG. 11C shows images of Oil Red O and H&E stained histologic sectionsof representative aortas from LDLR-KO mice fed a normal diet or fed ahigh cholesterol diet and administered at 12 weeks either theAAV2/8.eNOXpr-3xFLAG-mCherry vector or the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector.

FIG. 11D shows images of H&E stained histologic sections ofrepresentative aortas from LDLR-KO mice fed a normal diet or fed a highcholesterol diet and administered at 12 weeks either theAAV2/8.eNOXpr-3xFLAG-mCherry vector or the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector.

FIG. 12 shows high-resolution ultrasound imaging and systolic bloodvelocity in the aortic arch of LDLR-KO mice fed a normal diet or fed ahigh cholesterol diet and administered at 12 weeks either theAAV2/8.eNOXpr-3xFLAG-mCherry vector or the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector. (*P<0.05).

FIG. 13 shows high-resolution ultrasound imaging and systolic bloodvelocity in the abdominal region of the aorta of LDLR-KO mice fed anormal diet or fed a high cholesterol diet and administered at 12 weekseither the AAV2/8.eNOXpr-3xFLAG-mCherry vector or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector. *P<0.05.

FIG. 14 shows high-resolution ultrasound imaging and cross-sectionalarea of the thoracic region of the aortas of LDLR-KO mice fed a normaldiet or fed a high cholesterol diet and administered at 12 weeks eitherthe AAV2/8.eNOXpr-3xFLAG-mCherry vector or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector. *P<0.05.

FIG. 15 shows high-resolution ultrasound imaging and wall thickness ofthe aorta arch region of LDLR-KO mice fed a normal diet or fed a highcholesterol diet and administered at 12 weeks either theAAV2/8.eNOXpr-3xFLAG-mCherry vector or the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector. *P<0.05.

DESCRIPTION OF CERTAIN SEQUENCES

Table 1 provides a listing of certain sequences referenced herein.

TABLE 1 Description of Certain Sequences SEQ ID NO: SEQUENCE DESCRIPTION1 gttttccatatttaaaagtagtaaattggataccatacatgaa 5′ flanking sequenceaatcagctccaggtggattcaaaacataaatgtaaaatgcaaa of human NOX1aatataaaatttctagaagaaaatataaaagagtatcttgata genetctgggtagtgatggatttctaaaacaagacataaaatgcata Nucleotides 1-2027aatcataaaagaaatgactggtaatcagagtgcattaaaatta of GenBankagaacttccatttatcagaaaacactattaagagactgaaaag Accession No.acaagccataaacataagcaataaaagattagtataagattat DQ314883 .1aaacagaaccctaagaatctaaaagcaaaagaaaaaccaatagaaagatagaccaaaaagtagaataggctcagaataggctcttttaaaaagagaaaactcaaatggccagcagttgaattaaaagatgctcaaactcattagtaatcagggaaatgcaaattaaaatcataatacgatagttttccacacttacttgaattataaaaacaaaaaagtctggaaaataccaagggttggtaagcatgtagaggaagtagaactctcattcataactctctgtagtatacatttaggtggtcacttcggaacgggtttggaattacacagcaaagtagaatatgtgcaaatctcaggaccctggaattttactcctgggtatataccttagagaaactgtagcatatgtgtgacattcgatcaacattgttccatcatcatatccatcagtagtaggatgaatgaatacattaatgtatattcattcatgcaatggcatattagatagcagtgtaagtgaaccgcaattacatgtacatgtatgaatctcaaaaacccaatgttgaaagaagcaaaccacagaagcatacatacacactgccaggtttcatttacaaaaagttcaaaaacaggaaaaactaaacaatatattgcttagggatgcaattatagttagtaaaaatataaagaaaaataacagaatgattaccccaaatttcaggatagtgattacatccggtggggtagaggaggggaagaagatagatgtgatcagggagggaaatacaaagagctttaagatactggagaaaaatagtctattttctttaatctgagtagtgaacacatagatacttattccttaaaattattctttaagttacatatgtatgttttatatactcttctgtgtatatttcaccattttagaaaagggaaaaaaaatcagtgcccagagctgaacacacaactctagtaaatctatcatactagaagacaatcatctccattcttttgagtgctctgcctctgtttattttgaaccaaagtgcacttttatacttgttaaattttctcttgctctatttggcccttcttttcacttgtccttccagccagtcaagttctccccaaagccatcatcatatatgtcaaccacagatcatcctccaggggaactggtatgctaaagtttctgagctagccaggctgaaatccaaatggcagccggcagatgtggcaacagtttgaaaagtgcactttgaaacagcttccttaccacacacgcttccctccctacttctcctgaagtaatctgtttacagacccagactaataatcttttttatgagaaactttagcaaatcttttatctaggaaggcaatgcttcacattaggtcatgttgataagatgatgagagagaatattttcatccaagaatgttgctatttcctgaagcagtaaaatccccacaggtaaaacccttgtggttctcatagatagggctggtctatctaagctgatagcacagttctgtccagagaaggaaggcagaataaacttattcattcccaggaactcttggggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaatgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaaccacctc ttgaca 2tttgaaacagcttccttaccacacacgcttccctccctacttc Exemplary humantcctgaagtaatctgtttacagacccagactaataatcttttt NOX1 core promotertatgagaaactttagcaaatcttttatctaggaaggcaatgcttcacattaggtcatgttgataagatgatgagagagaatattttcatccaagaatgttgctatttcctgaagcagtaaaatccccacaggtaaaacccttgtggttctcatagatagggctggtctatctaagctgatagcacagttctgtccagagaaggaaggcagaataaacttattcattcccaggaactcttggggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaatgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaaccacctcttgaca 3tttgaaacagcttccttaccacacacgcttccctccctacttc Exemplary varianttcctgaagtaatctgtttacagacccagactaataatcttttt human NOX1 coretatgagaaactttagcaaatcttttatctaggaagg c caatgc promoterttcacattaggtcatgttgataagatgatgagagagaatattttcatccaagaatgttgctatttcctgaagcagtaaaatccccacaggtaaaacccttgtggttctcatagatagggctggtctatctaagctgatagcacagttctgtccagagaaggaaggcagaataaacttattcattcccaggaactcttggggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaa t tgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaaccacctcttgaca tgaac gcgtgccacc 4 gaggaggggattccc aagatcgagga ggggattccc aagatcg Exemplary variant aggaggggattccc aagatc aaaagt atgcaaat ccctgaaaa NOX1 core promoter agtatgcaaat ccctgatttgaaacagcttccttaccacacac with three NF-κBgcttccctccctacttctcctgaagtaatctgtttacagaccc binding sequencesagactaataatcttttttatgagaaactttagcaaatctttta (italic, underlined)tctaggaaggccaatgcttcacattaggtcatgttgataagat and two Oct1 bindinggatgagagagaatattttcatccaagaatgttgctatttcctg sequences (bold,aagcagtaaaatccccacaggtaaaacccttgtggttctcata underlined) at the 5′gatagggctggtctatctaagctgatagcacagttctgtccag endagaaggaaggcagaataaacttattcattcccaggaactcttg “eNOX1 promoter”gggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaattgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaac cacctcttgacatgaacgcgtgccacc5 gggrnyyycc, wherein r is a purine, y is a Exemplary consensuspyrimidine, and n is any nucleotide sequence of an NF- κB binding site 6ggrrnnyycc, wherein r is a purine, y is a Exemplary consensuspyrimidine, and n is any nucleotide sequence of an NF- κB binding site 7rggrnnhhyyb; wherein r is a purine; y is a Exemplary consensuspyrimidine; h is an adenine, a cytosine, or sequence of an NF-a thymine; b is a guanine, thymine, or κB binding sitecytosine; and n is any nucleotide 8ggggatyccc, wherein y is a pyrimidine Exemplary consensussequence of an NF- κB binding site 9gggrntttcc, wherein n is any nucleotide Exemplary consensussequence of an NF- κB binding site 10nggnnwttcc, wherein w is an adenine or a Exemplary consensusthymine; and n is any nucleotide sequence of an NF- κB binding site 11gggrnnyycc, wherein r is a purine, y is a Exemplary consensuspyrimidine, and n is any nucleotide sequence of an NF- κB binding site12 ggggaatcccc Exemplary sequence of an NF-κB binding site 13ggggactttcc Exemplary sequence of an NF-κB binding site 14 agggggatctgExemplary sequence of an NF-κB binding site 15 agggaagttaExemplary sequence of an NF-κB binding site 16 ctggggatttaExemplary sequence of an NF-κB binding site 17 gggaattcccExemplary sequence of an NF-κB binding site 18 gggaatttccExemplary sequence of an NF-κB binding site 19 ggggattcccExemplary sequence of an NF-κB binding site 20 gaggaggggattcccaagatcExemplary sequence of an NF-κKB binding site 21 atgcaaatExemplary sequence of an Oct1 binding site 22gaggaggggattcccaagatcgaggaggggattcccaagatcg eNOXpr-3xFLAG-aggaggggattcccaagatcaaaagtatgcaaatccctgaaaa mCherryagtatgcaaatccctgatttgaaacagcttccttaccacacacgcttccctccctacttctcctgaagtaatctgtttacagacccagactaataatcttttttatgagaaactttagcaaatcttttatctaggaaggccaatgcttcacattaggtcatgttgataagatgatgagagagaatattttcatccaagaatgttgctatttcctgaagcagtaaaatccccacaggtaaaacccttgtggttctcatagatagggctggtctatctaagctgatagcacagttctgtccagagaaggaaggcagaataaacttattcattcccaggaactcttggggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaattgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaaccacctcttgacatgaacgcgtgccaccatgtctgactataaagaccatgatggggactacaaagaccatgatatagattacaaagacgatgatgacaaaatggttagcaagggggaggaagacaatatggccataattaaagaattcatgcgcttcaaagttcacatggaaggaagcgtgaacggacatgagttcgagatagaaggcgagggcgaggggcggccctatgagggaacgcagactgctaaactgaaggttactaaaggtggccctcttcctttcgcatgggacatcctgtctccgcagttcatgtatggatccaaggcatatgttaagcatccggctgatataccagattacctcaaattgagctttcctgaagggtttaagtgggaaagggtcatgaactttgaagacggtggagttgtgacagttacacaggattcatcacttcaggacggtgagtttatatacaaggttaaacttaggggaactaattttccttccgacggccccgtcatgcagaaaaaaaccatggggtgggaggcgagctccgagcggatgtacccagaggatggagcactgaagggcgaaataaaacagcgactgaaattgaaagacggaggtcactatgatgcagaagttaagacgacatacaaggccaaaaagccagttcagttgccgggtgcatataacgtcaatatcaagctggacattacatcccacaatgaggattatacgatagtggagcagtatgagcgggcagaagggcggcactccacaggaggaatggacgaactctataaatgacccaccagccttgtcctaataaaattaagttgcatcattttgtttgacta ctcgagcccctgca 23MSDYKDHDGDYKDHDIDYKDDDDKMVSKGEEDNMAIIKEFMRF 3xFLAG-mCherryKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFA amino acid sequenceWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGINFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDEL YK 24MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGG Exemplary wildtypeTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARL human FoxP3 aminoGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMI acid sequenceSLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFP NCBI Ref Sequence:NPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL NP_054728.2KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPG P 25EKGRAQCLLQREMVQSLEQQLVLEKEKLSAPIQAHLAGK Exemplary humanFOXP3 zinc finger and leucine zipper region 26MMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQLQQL Exemplary wildtypeWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPH human FoxP1ASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVC sequenceEDFQSFLKHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKE GenBank:RLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSL AF146698.2PHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSADIAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRMFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEFQKRRPQKISGNPSLIKNMQSSHAYCTPLNAALQASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDF DHDRDYEDEPVNEDME 27DRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVK Exemplary human FOXP1 zinc fingerand leucine zipper region 28 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGExemplary TFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARL FOXP3(P1) aminoGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMI acid sequenceSLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFP FOXP1 zinc fingerNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL and leucine zipperKHCQADHLLDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMT region underlinedHLHVKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPG P 29atgccgaatccccggccaggcaagcccagtgccccgtcacttg Exemplarycccttgggcctagtcctggggcttcaccatcctggcgagctgc FoxP3(P1) nucleotideacctaaggcatctgacctcttgggggcacgaggaccgggcggg sequenceacgtttcagggaagggaccttagaggcggagctcatgcaagct FoxP1 zinc fingercttcttcactgaacccgatgccgccgagtcagttgcaactccc and leucine zippercacactcccactcgtaatggtggcgccctctggcgcaagactc region underlinedggacctctcccacacctgcaagccctcttgcaggacagaccacacttcatgcaccaactttcaacggttgacgcacacgcacggacaccagtgctgcaagttcatccacttgaatcccctgccatgatcagcctgacaccgcctactaccgcgacaggtgtcttttctttgaaagcgaggcctggattgccacctggcatcaatgtggcgtccctggagtgggtttcccgcgaacctgctctcctgtgcacatttccaaacccgagtgcgccgcgaaaagatagtacgttgtccgcagtacctcagagctcatatccacttttggcaaacggtgtgtgtaaatggcctggatgcgaaaaagtattcgaagagccggaggactttttgaaacattgccaagctgaccacctgctcgatgatcggtcaaccgcgcaatgcagggtgcaaatgcaagttgtacaacagctcgaattgcagttggcgaaggacaaggagaggctgcaagcaatgatgacccatcttcatgttaaaatggccctgaccaaggcaagctctgttgcaagctccgacaaaggctcttgctgtatcgtagcggcgggatctcaaggaccggtcgtcccagcgtggagtggccctcgggaagcccctgatagtcttttcgccgtgagacgccacctgtggggcagccatggaaactccacttttcctgaatttttgcacaatatggactactttaagttccataacatgcgccccccgtttacatacgcgacgctcatccggtgggcaatcttggaagcgcctgaaaaacaacgaaccttgaacgagatatatcattggttcacgcgaatgttcgctttcttcagaaatcacccggctacttggaagaatgccataagacacaatctttctctccataaatgctttgtaagggtcgagtccgaaaaaggggcagtatggactgttgacgagctggagtttcggaaaaagcggtcacaacgcccgtcaagatgctcaaaccctaccccaggc ccttga 30ttagataaaggctgtctccgcgcctatataaaactcttgtttt Exemplary mini-tcttttttctctatcagttcatttgtagcatcttaatttacta promoter TATA boxtccttctactatcagttgccgccgccgtcgacgccacc nucleotide sequence 31MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRD Exemplary humanLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEM IL10 amino acidIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRF sequenceLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEEDIFINYIEAYM UniProtKB TMKIRNAccession No. P22301 32 atgcactcttctgcacttctgtgctgcctcgtgctcctgacagExemplary human gtgtcagggcgagtcccggtcagggtacgcaatctgaaaactcIL10 nucleotide ctgcacccactttccggggaatttgcccaacatgctgagggat sequencectgagagacgctttcagccgcgttaagacattcttccagatgaaagatcagctcgataatcttctgttgaaagagtcactgcttgaggattttaaagggtatttggggtgccaggctctgtcagaaatgatacagttctatctcgaagaggtgatgcctcaagcggagaaccaagatccagacataaaggctcacgttaattccttgggcgagaatctgaaaaccctgaggcttaggctgagacgctgtcatcgcttcttgccctgtgaaaacaaatccaaagcggtagagcaggtcaaaaatgcctttaataagctgcaagagaaggggatatataaggcaatgtctgagtttgatatctttataaactatatagaagcttacatg acaatgaaaattcggaattag 33ttaattgaggggccgggctcgagtgcctaataaaaaacattta Exemplary 5′ttttcattgccccctgcagaagctttaaaccggttatcgataa sequence including tcaacctcpolyadenylation (poly A) sequence 34ggggttcctgcggccgcacgcgtctgcagcccatgcatgagga Exemplaryggggattcccaagatcgaggaggggattcccaagatcgaggag eNOX1pr-gggattcccaagatcaaaagtatgcaaatccctgaaaaagtat FoxP3(P1)-IL10gcaaatccctgatttgaaacagcttccttaccacacacgcttc nucleotide sequencecctccctacttctcctgaagtaatctgtttacagacccagactaataatcttttttatgagaaactttagcaaatcttttatctaggaaggccaatgcttcacattaggtcatgttgataagatgatgagagagaatattttcatccaagaatgttgctatttcctgaagcagtaaaatccccacaggtaaaacccttgtggttctcatagatagggctggtctatctaagctgatagcacagttctgtccagagaaggaaggcagaataaacttattcattcccaggaactcttggggtaggtgtgtgtttttcacatcttaaaggctcacagaccctgcgctggacaaattgttccattcctgaaggacctctccagaatccggattgctgaatcttccctgttgcctagaagggctccaaaccacctcttgacatgaacgcgtgccaccatgccgaatccccggccaggcaagcccagtgccccgtcacttgcccttgggcctagtcctggggcttcaccatcctggcgagctgcacctaaggcatctgacctcttgggggcacgaggaccgggcgggacgtttcagggaagggaccttagaggcggagctcatgcaagctcttcttcactgaacccgatgccgccgagtcagttgcaactccccacactcccactcgtaatggtggcgccctctggcgcaagactcggacctctcccacacctgcaagccctcttgcaggacagaccacacttcatgcaccaactttcaacggttgacgcacacgcacggacaccagtgctgcaagttcatccacttgaatcccctgccatgatcagcctgacaccgcctactaccgcgacaggtgtcttttctttgaaagcgaggcctggattgccacctggcatcaatgtggcgtccctggagtgggtttcccgcgaacctgctctcctgtgcacatttccaaacccgagtgcgccgcgaaaagatagtacgttgtccgcagtacctcagagctcatatccacttttggcaaacggtgtgtgtaaatggcctggatgcgaaaaagtattcgaagagccggaggactttttgaaacattgccaagctgaccacctgctcgatgatcggtcaaccgcgcaatgcagggtgcaaatgcaagttgtacaacagctcgaattgcagttggcgaaggacaaggagaggctgcaagcaatgatgacccatcttcatgttaaaatggccctgaccaaggcaagctctgttgcaagctccgacaaaggctcttgctgtatcgtagcggcgggatctcaaggaccggtcgtcccagcgtggagtggccctcgggaagcccctgatagtcttttcgccgtgagacgccacctgtggggcagccatggaaactccacttttcctgaatttttgcacaatatggactactttaagttccataacatgcgccccccgtttacatacgcgacgctcatccggtgggcaatcttggaagcgcctgaaaaacaacgaaccttgaacgagatatatcattggttcacgcgaatgttcgctttcttcagaaatcacccggctacttggaagaatgccataagacacaatctttctctccataaatgctttgtaagggtcgagtccgaaaaaggggcagtatggactgttgacgagctggagtttcggaaaaagcggtcacaacgcccgtcaagatgctcaaaccctaccccaggcccttgattagataaaggctgtctccgcgcctatataaaactcttgtttttcttttttctctatcagttcatttgtagcatcttaatttactatccttctactatcagttgccgccgccgtcgacgccaccatgcactcttctgcacttctgtgctgcctcgtgctcctgacaggtgtcagggcgagtcccggtcagggtacgcaatctgaaaactcctgcacccactttccggggaatttgcccaacatgctgagggatctgagagacgctttcagccgcgttaagacattcttccagatgaaagatcagctcgataatcttctgttgaaagagtcactgcttgaggattttaaagggtatttggggtgccaggctctgtcagaaatgatacagttctatctcgaagaggtgatgcctcaagcggagaaccaagatccagacataaaggctcacgttaattccttgggcgagaatctgaaaaccctgaggcttaggctgagacgctgtcatcgcttcttgccctgtgaaaacaaatccaaagcggtagagcaggtcaaaaatgcctttaataagctgcaagagaaggggatatataaggcaatgtctgagtttgatatctttataaactatatagaagcttacatgacaatgaaaattcggaattagttaattgaggggccgggctcgagtgcctaataaaaaacatttattttcattgccccctgcagaagctttaaaccggttatcgata atcaacctc 35MMQESATETISNSSMNQNGMSTLSSQLDAGSRDGRSSGDTSSE Exemplary wildtypeVSTVELLHLQQQQALQAARQLLLQQQTSGLKSPKSSDKQRPLQ human FoxP2VPVSVAMMTPQVITPQQMQQILQQQVLSPQQLQALLQQQQAVM sequenceLQQQQLQEFYKKQQEQLHLQLLQQQQQQQQQQQQQQQQQQQQQ NCBI Ref No.QQQQQQQQQQQQQQQQQQQHPGKQAKEQQQQQQQQQQLAAQQL NP_055306.1VFQQQLLQMQQLQQQQHLLSLQRQGLISIPPGQAALPVQSLPQAGLSPAEIQQLWKEVTGVHSMEDNGIKHGGLDLTTNNSSSTTSSNTSKASPPITHHSIVNGQSSVLSARRDSSSHEETGASHTLYGHGVCKWPGCESICEDFGQFLKHLNNEHALDDRSTAQCRVQMQVVQQLEIQLSKERERLQAMMTHLHMRPSEPKPSPKPLNLVSSVTMSKNMLETSPQSLPQTPTTPTAPVTPITQGPSVITPASVPNVGAIRRRHSDKYNIPMSSEIAPNYEFYKNADVRPPFTYATLIRQAIMESSDRQLTLNETYSWFTRTFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEYQKRRSQKITGSPTLVKNIPTSLGYGAALNASLQAALAESSLPLLSNPGLINNASSGLLQAVHEDLNGSLDHIDSNGNSSPGCSPQPHIHSIHVKEEPVIAEDEDCPMS LVTTANHSPELEDDREIEEEPLSEDLE36 MMVESASETIRSAPSGQNGVGSLSGQADGSSGGATGTTASGTG Exemplary wildtypeREVTTGADSNGEMSPAELLHFQQQQALQVARQFLLQQASGLSS human FoxP4PGNNDSKQSASAVQVPVSVAMMSPQMLTPQQMQQILSPPQLQA sequenceLLQQQQALMLQQEYYKKQQEQLHLQLLTQQQAGKPQPKEALGN GenBank:KQLAFQQQLLQMQQLQQQHLLNLQRQGLVSLQPNQASGPLQTL KJ900035.1PQAAVCPTDLPQLWKGEGAPGQPAEDSVKQEGLDLTGTAATATSFAAPPKVSPPLSHHTLPNGQPTVLTSRRDSSSHEETPGSHPLYGHGECKWPGCETLCEDLGQFIKHLNTEHALDDRSTAQCRVQMQVVQQLEIQLAKESERLQAMMAHLHMRPSEPKPFSQPLNPVPGSSSFSKVTVSAADSFPDGLVHPPTSAAAPVTPLRPPGLGSASLHGGGPARRRSSDKFCSPISSELAQNHEFYKNADVRPPFTYASLIRQAILETPDRQLTLNEIYNWFTRMFAYFRRNTATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEREYQKRRPPKMTGSPTLVKNMISGLSYGALNASYQAALAESSFPLLNSPGMLNPGSASSLLPLSHDDVGAPVEPLPSNGSSSPPRLSPPQYSHQVQVKEEPAEAEEDRQPGPPLGAPNPSASGPPEDRDLEEELPGEELS 37 aaagatagtacgttgtccgcagFoxP3(P1) forward primer 38 atttgcaccctgcattgcgc FoxP3(P1) reverseprimer 39 tctgtgctgcctcgtgctcc IL10 forward primer 40tctgacagagcctggcaccc IL10 reverse primer 41 tccactcacggcaaattcaacmGAPDH forward primer 42 cgctcctggaagatggtgatg mGAPDH reverse primer

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

The present disclosure provides NOX1 core promoters for regulatingexpression of transgenes in response to vascular pathology. In someembodiments, a NOX1 core promoter may express a transgene in ahigh-lipid environment or in the presence of factors associated withinflammation, such as Angiotensin II (Ang II), Lipopolysaccharides(LPS), or Oxidized Low Density Lipoprotein (Ox-LDL). A NOX1 corepromoter of reduced size, such as a NOX1 core promoter comprising lessthan 600 nucleotides, allows for larger or multiple transgenes to beused in a single gene therapy vector having limited packaging capacity,such as AAV. For example, NOX1 core promoters within nucleotidepositions 1561 and 1817 of SEQ ID NO: 1 are provided. A NOX1 corepromoter may be modified, such as by removing an unnecessary ATG startsite and/or adding a TATA box and/or a CAAT box. One or moretranscription factor binding sites may also be added to an isolatednucleic acid molecule comprising a NOX1 core promoter, for example, atthe 5′ end of a NOX1 core promoter. NOX1 core promoters described hereinmay be useful in gene therapy methods for regulated expression oftherapeutic proteins, such as anti-inflammatory molecules, for thetreatment of vascular and/or inflammation-associated diseases.

The present disclosure further provides variant FOXP polypeptides havinga zinc finger and leucine zipper region of a different FOXP polypeptide.Such variant FOXP polypeptides may preferentially dimerize with eachother over the corresponding endogenous wildtype FOXP polypeptide. Forexample, a variant FOXP3 polypeptide having a zinc finger and leucinezipper region of FOXP1 (SEQ ID NO: 28) was designed and expressed.Variant FOXP3 polypeptides described herein may be useful for thetreatment of vascular and/or inflammation-associated diseases.

Nucleic acids, vectors, and expression systems comprising a NOX1 corepromoter and/or encoding variant FOXP3 polypeptides are also described.Further methods of expressing variant FOXP3 polypeptides, includingregulated expression, by gene therapy methods are described. Forexample, several in vitro and in vivo animal studies are describedinvolving transduction with AAV vectors delivering an exemplary NOX1core promoter regulating expression of a reporter protein or multipletherapeutic proteins (e.g., an exemplary variant FOXP3 polypeptide andIL10).

An in vivo animal model of established and ongoing atherosclerosis fortesting treatment options that more closely mimics the human clinicalsituation is also described. In some embodiments, low densitylipoprotein receptor knockout (LDLR-KO) mice or rats are fed a highcholesterol diet (HCD) for a period of time, such as at least 56 days,before administering a therapeutic agent (e.g., a gene therapy vectorexpressing a therapeutic protein). An AAV vector delivering an exemplaryNOX1 core promoter regulating expression of a variant FOXP3 polypeptideand IL10 was tested using this animal model. LDLR-KO mice maintained ona HCD and administered the eNOX1-FOXP3(P1)-IL10 vector at week 12 showedsignificantly less vascular pathology when analyzed at week 20 byhigh-resolution ultrasound imaging compared to mice maintained on a HCDand administered a control vector.

For the convenience of the reader, the following definitions of termsused herein are provided.

As used herein, numerical terms are calculated based upon scientificmeasurements and, thus, are subject to appropriate measurement error. Insome instances, a numerical term may include numerical values that arerounded to the nearest significant figure.

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise specified. As used herein, the term “or” means “and/or” unlessspecified otherwise. In the context of a multiple dependent claim, theuse of “or” when referring back to other claims refers to those claimsin the alternative only.

Exemplary Nucleic Acid Molecules and Polypeptides

“Nucleic acid molecule” or “polynucleotide” are used interchangeablyherein to refer to a polymer of nucleotides. A nucleotide is composed ofa base, specifically a purine or pyrimidine base (i.e., cytosine (C),guanine (G), adenine (A), thymine (T) or uracil (U)); a sugar (i.e.,deoxyribose or ribose); and a phosphate group. A nucleic acid moleculemay be described by the nucleotide sequence representing its primarylinear structure. A nucleotide sequence is typically represented from 5′to 3′. Nucleic acid molecules include, for example, deoxyribonucleicacid (DNA) including genomic DNA, mitochondrial DNA, methylated DNA, andthe like; ribonucleic acid (RNA), including messenger RNA (mRNA), smallinterfering RNA (siRNA), microRNA (miRNA), non-coding RNAs. A nucleicacid molecule can be single-stranded or double-stranded DNA or RNA.Alternatively, a nucleic acid molecule may be a DNA-RNA duplex.

A nucleotide base may be represented using the International Union ofPure and Applied Chemistry (IUPAC) nucleotide code shown in Table 2.

TABLE 2 IUPAC nucleotide code Base A Adenine C Cytosine G Guanine T (orU) Thymine (or Uracil) R A or G Y C or T S G or C W A or T K G or T M Aor C B C or G or T D A or G or T H A or C or T V A or C or G N any base

A nucleic acid molecule or polypeptide described herein may be from anysource unless otherwise indicated. The source may be a vertebratesource, including mammals such as primates (e.g., humans or cynomolgusmonkeys), rodents (e.g., mice and rats), etc.

“Heterologous” as used herein, refers to a nucleic acid molecule orpolypeptide that is foreign to its surrounding nucleic acid molecule orpolypeptide.

“Promoter,” as used herein, refers to a nucleic acid molecule comprisinga nucleotide sequence upstream of a transcription start site that iscapable of initiating transcription. A promoter may be a constitutivepromoter or a regulated promoter.

A “constitutive promoter” is an unregulated promoter that allows forcontinual transcription. Non-limiting exemplary constitutive promotersinclude cytomegalovirus (CMV) immediate early promoter, simian virus 40(SV40) early promoter, adenovirus major late promoter (MLP), Roussarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter,phosphoglycerate kinase 1 (PGK) promoter, elongation factor-alpha (EF1a)promoter, ubiquitin promoters, actin promoters (such as human β-actinpromoter and chicken β-actin promoter), chicken β-actin promoter and CMVearly enhancer, tubulin promoters, immunoglobulin promoters, afunctional fragment thereof, or a combination of any of the foregoing.In some embodiments, the promoter may be a CMV promoter. In someembodiments, the promoter may be a truncated CMV promoter. In otherembodiments, the promoter may be an EF1a promoter.

Non-limiting exemplary regulated promoters include those inducible by orresponsive to heat shock, light, chemicals, lipids, peptides, metals,steroids, antibiotics, or alcohol. A regulated promoter may be apathology-specific promoter and/or a tissue-specific promoter. In someembodiments, a regulated promoter may be one that regulates expressionof a transgene in the presence of one or more factors associated withinflammation, such as Ang II, LPS, Ox-LDL and/or carbamylated LDL. Insome embodiments, the promoter is responsive to sheer stress. In someembodiments, the promoter is responsive to dyslipidemia.

A “pathology-specific promoter,” as used herein, is a type of regulatedpromoter that is generally responsive to stimuli characteristic of adisease, condition, or disorder being treated. In some embodiments, apathology-specific promoter may preferentially express a transgene inthe presence of a high-lipid environment characteristic of a vascularpathology.

A “tissue-specific promoter,” as used herein, is a type of regulatedpromoter that is generally responsive to stimuli characteristic of oneor more cell types. In some embodiments, a tissue-specific promoter maypreferentially express a transgene in liver cells. In some embodiments,a tissue-specific promoter may preferentially express a transgene inmuscle cells. In some embodiments, a tissue-specific promoter maypreferentially express a transgene in endothelial cells, such asvascular endothelial cells.

“Core promoter,” as used herein, refers to a nucleic acid moleculecomprising a smaller portion of a promoter nucleotide sequence that iscapable of initiating transcription.

In some embodiments, a promoter or a core promoter comprises one or moretranscription factor binding sites, a TATA box or TATA-like box, and/orone or more enhancer elements.

“Transcription factor binding site” or “binding site” as usedinterchangeably herein, refers to the region of a nucleic acid moleculeto which a transcription factor binds. A transcription factor bindingsite may be described by the nucleotide sequence (e.g., a specificsequence or a consensus sequence) representing its primary linearstructure.

“Consensus sequence,” as used herein, refers to a sequence of frequentresidues identified by sequence alignment of related sequences.

“NOX1 promoter,” as used herein, refers to a nucleic acid moleculecomprising a nucleotide sequence upstream of a transcription start siteof a NADPH Oxidase 1 (NOX1) coding sequence from any source. The sourcemay be a vertebrate source, including mammals such as primates (e.g.,humans or cynomolgus monkeys), rodents (e.g., mice and rats), etc. Forexample, a human NOX1 promoter may comprise the nucleotide sequence ofSEQ ID NO: 1 (a 2027 nucleotide sequence upstream of the ATG start siteof the human NOX1 gene reported as GenBank Accession No. DQ314883.1).

“NOX1 core promoter,” as used herein, refers to a smaller portion of aNOX1 promoter or variant thereof that is capable of initiatingtranscription. For example, a NOX1 core promoter may be less than 600nucleotides, less than 550 nucleotides, less than 500 nucleotides, lessthan 480 nucleotides, or less than 470 nucleotides. In some embodiments,a NOX1 core promoter comprises a nucleotide sequence within nucleotidepositions 1561 and 1817 of SEQ ID NO: 1.

“Variant NOX1 core promoter,” as used herein, refers to a NOX1 corepromoter that differs from a reference NOX1 core promoter by a single ormultiple non-native nucleotide substitutions, deletions, and/oradditions and retains the ability to initiate transcription. Forexample, a variant NOX1 core promoter may have one or more nucleotidesubstitutions, deletions, and/or additions that knocks out anunnecessary ATG start site, that adds a stop codon (e.g., TGA, TAA, orTAG), that adds a Kozak consensus sequence, and/or that adds a CAAT box.

A “point mutation,” as used herein, refers to a mutation that involves asingle nucleotide base or very few nucleotide bases of a nucleic acidmolecule. For example, the mutation may be the loss of one base,substitution of one nucleotide base for another, or the insertion of anadditional nucleotide base.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a nucleic acid molecule or a polypeptide aredefined as the percentage of nucleotides or amino acid residues in acandidate sequence that are identical with the nucleotides or amino acidresidues in the reference nucleic acid molecule or polypeptide, afteraligning the sequences and introducing gaps, if necessary to achieve themaximum percent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of sequences beingcompared.

In some embodiments, a nucleic acid molecule or a polypeptide has atleast about 50% sequence identity with the reference nucleic acidmolecule or polypeptide after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. A nucleic acid molecule may include, for instance,addition or deletion of one or more nucleic acid bases at the 5′ or 3′terminus compared to a reference nucleic acid molecule. A polypeptidemay include, for example, addition or deletion at the N- or C-terminusof the polypeptide compared to a reference polypeptide. In someembodiments, a nucleic acid molecule or a polypeptide has at least about50% sequence identity, at least about 60% sequence identity, at leastabout 65% sequence identity, at least about 70% sequence identity, atleast about 75% sequence identity, at least about 80% sequence identity,at least about 85% sequence identity, at least about 90% sequenceidentity, at least about 91% sequence identity, at least about 92%sequence identity, at least about 93% sequence identity, at least about94% sequence identity, at least about 95% sequence identity, at leastabout 96% sequence identity, at least about 97% sequence identity, atleast about 98% sequence identity, at least about 99% sequence identity,or 100% sequence identity with the sequence of a reference nucleic acidmolecule or polypeptide.

In some embodiments, a NOX1 core promoter comprises a nucleotidesequence having at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100% sequenceidentity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, a NOX1core promoter comprises the nucleotide sequence of SEQ ID NO: 2 or SEQID NO: 3.

A NOX1 promoter or a NOX1 core promoter may naturally comprise one ormore endogenous transcription factor binding sites.

In some embodiments, a NOX1 core promoter is modified to contain one ormore heterologous transcription factor binding sites (e.g., one or moreNFκB binding sites and/or one or more Oct1 binding sites) within thecore promoter sequence. In some embodiments, a nucleic acid moleculecomprising a NOX1 core promoter may further comprise at least oneheterologous transcription factor binding site positioned at the 5′ endor the 3′ end of the NOX1 core promoter. For example, a nucleic acidmolecule comprising a NOX1 core promoter may further comprise at leastone NFκB binding site, which may be positioned at the 5′ end or the 3′end of the NOX1 core promoter. In some embodiments, a nucleic acidmolecule comprising a NOX1 core promoter may further comprise at leastone Oct1 binding site, which may be positioned at the 5′ end or the 3′end of the NOX1 core promoter.

In some embodiments, an NFκB binding site may be described by a specificsequence, such as, including but not limited to, the nucleotide sequenceof SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.An NFκB binding site may be described by a consensus sequence, such as,including but not limited to, the nucleotide sequence of SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,or SEQ ID NO: 11. Exemplary NFκB binding sites are described in Wan Fand Lenardo M, 2009; Wong D, et al., 2011; Kunsch C, et al., 1992;Udalova I A, et al., 2002; and Takano T and Cybulsky A V, 2000, each ofwhich is incorporated herein by reference in their entirety.

In some embodiments, an Oct1 binding site may be described by thenucleotide sequence of SEQ ID NO: 21. See Jenuwein T and Grosschedl R,1991; Chen J, BCJ, 2006; Zhao F Q, et al., 2013, each of which isincorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a NOX1 corepromoter comprises at least one, at least two, at least three, at leastfour, or at least five heterologous NFκB binding sites. In someembodiments, a nucleic acid molecule comprising a NOX1 core promotercomprises at least one, at least two, at least three, at least four, orat least five heterologous Oct1 binding sites. In some embodiments, avariant NOX1 core promoter comprises at least one, at least one, atleast two, at least three, at least four, or at least five heterologousNFκB binding sites. In some embodiments, a variant NOX1 core promotercomprises at least one, at least one, at least two, at least three, atleast four, or at least five heterologous Oct1 binding sites.

In some embodiments, a nucleic acid molecule comprises a nucleotidesequence having at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100% sequenceidentity to SEQ ID NO: 4. In some embodiments, a nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO: 4.

“Transgene,” as used herein refers to a nucleic acid molecule thatencodes a product that may be useful in biotechnology and medicine, suchas proteins and RNA. A transgene is generally represented by itsnucleotide coding sequence. Exemplary proteins include enzymes,cytokines, receptors, and reporter proteins, etc. Exemplary RNA includesort hairpin RNA (shRNA), transfer RNA (tRNA), double stranded (dsRNA),ribosomal RNA, catalytic RNA, antisense RNA, messenger RNA (mRNA), smallinterfering RNA (siRNA), microRNA (miRNA), and non-coding RNAs.

A nucleotide coding sequence of a transgene can be operatively linked toregulatory components in a manner which permits transgene transcription,translation, and/or expression in a target cell. In some embodiments,the regulatory component comprises a NOX1 promoter, a NOX1 corepromoter, or a variant NOX1 core promoter, as described herein. In someembodiments, an isolated nucleic acid molecule comprises a NOX1 corepromoter or a variant NOX1 core promoter and a nucleotide sequenceencoding a transgene, such as a reporter protein or a therapeuticpolypeptide (e.g., an anti-inflammatory molecule).

In some embodiments, a transgene comprises a nucleotide coding sequencefor a reporter protein, such as a FLAG-tag, a β-lactamase, aβ-galactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, agreen fluorescent protein (GFP), a red fluorescent protein (RFP), suchas mCherry, chloramphenicol acetyltransferase (CAT), luciferase,membrane bound proteins (e.g., CD2, CD4, CD8, the influenzahemagglutinin protein), and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins thereof. In some embodiments, atransgene comprises a nucleotide coding sequence for a FLAG-tag andmCherry fusion protein.

Such reporter proteins can provide signals detectable by conventionalmeans, including enzymatic, radiographic, colorimetric, fluorescence orother spectrographic assays, fluorescent activating cell sorting (FACS)assays and immunological assays, including enzyme linked immunosorbentassay (ELISA), radioimmunoassay (MA) and immunohistochemistry. Forexample, where the marker sequence is the LacZ gene, the presence of thevector carrying the signal is detected by assays for beta-galactosidaseactivity. Where the reporter protein is mCherry or luciferase, the cellexpressing the reporter protein may be measured visually by color orlight production in a luminometer.

In some embodiments, the transgene comprises a nucleotide codingsequence for a therapeutic polypeptide. In some embodiments, atherapeutic polypeptide is an anti-inflammatory molecule.

“Amino acid sequence,” means a sequence of amino acids residues in apolypeptide or protein. The terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues, and arenot limited to a minimum length. Such polymers of amino acid residuesmay contain natural or non-natural amino acid residues, and include, butare not limited to, peptides, oligopeptides, dimers, trimers, andmultimers of amino acid residues. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, phosphorylation, and the like.Furthermore, for purposes of the present disclosure, a “polypeptide”refers to a protein which includes modifications, such as deletions,additions, and substitutions (generally conservative in nature), to thenative sequence, as long as the protein maintains the desired activity.These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the proteins or errors due to PCR amplification.

An “amino acid substitution” refers to the replacement of one amino acidin a polypeptide with another amino acid. In some embodiments, an aminoacid substitution is a conservative substitution. Nonlimiting exemplaryconservative amino acid substitutions are shown in Table 3. Amino acidsubstitutions may be introduced into a molecule of interest and theproducts screened for a desired activity, for example, retained/improvedantigen binding, decreased immunogenicity, improved recombinantproduction, and/or enhanced pharmacokinetics.

TABLE 3 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp; Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes with another class.

“Wildtype polypeptide,” as used herein, refers to a non-mutated versionof a polypeptide that occurs in nature, or a fragment thereof. Awildtype polypeptide may be produced recombinantly.

“Variant polypeptide,” as used herein, refers to a polypeptide thatdiffers from a reference polypeptide by a single or multiple non-nativeamino acid substitutions, deletions, and/or additions. In someembodiments, a variant polypeptide retains at least one biologicalactivity of the reference polypeptide (e.g., a corresponding wildtypepolypeptide). A variant polypeptide includes, for instance, polypeptideswherein one or more amino acid residues are added, deleted, at the N- orC-terminus of the polypeptide.

“FOXP” and “FOXP polypeptide,” as used interchangeably herein, refer toa polypeptide comprising the entirety or a fragment of a protein fromthe Forkhead box protein family (such as FOXP1, FOXP2, FOXP3, or FOXP4)from any vertebrate source, including mammals such as primates (e.g.,humans and cynomolgus monkeys), rodents (e.g., mice and rats), andcompanion animals (e.g., dogs, cats, and equine), unless otherwiseindicated. FOXP includes variant FOXP polypeptides that substantiallyretain at least one biological activity of a wildtype FOXP polypeptide.

A “variant FOXP polypeptide” as used herein is a FOXP polypeptide thatdiffers from a reference FOXP polypeptide by single or multiple aminoacid substitutions, deletions, and/or additions and substantiallyretains at least one biological activity of the reference FOXP3polypeptide.

In some embodiments, FOXP may be a wildtype FOXP1, FOXP2, FOXP3, orFOXP4 polypeptide. An exemplary wildtype human FOXP1 comprises the aminoacid of SEQ ID NO: 26. An exemplary wildtype human FOXP2 comprises theamino acid of SEQ ID NO: 35. An exemplary wildtype human FOXP3 comprisesthe amino acid of SEQ ID NO: 24. An exemplary wildtype human FOXP4comprises the amino acid of SEQ ID NO: 36.

Wildtype FOXP polypeptides have a zinc finger and leucine zipper regionunderstood to be involved in protein dimerization. Kim J, et al., 2019;Wang B, et al., 2003; Song Z, et al., 2012. “Zinc finger and leucinezipper region,” as used herein, refers to a region of a polypeptidecomprising a zinc finger motif and a leucine zipper motif. In someembodiments, a zinc finger and leucine zipper region of a FOXP3polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In someembodiments, a zinc finger and leucine zipper region of a FOXP3polypeptide comprises an amino acid sequence within amino acid positions225 and 264 of SEQ ID NO: 24. In some embodiments, a zinc finger andleucine zipper region of a FOXP1 polypeptide comprises the amino acidsequence of SEQ ID NO: 27. In some embodiments, a zinc finger andleucine zipper region of a FOXP1 polypeptide comprises an amino acidsequence within amino acid positions 146 and 185 of SEQ ID NO: 26.

In some embodiments, a variant FOXP polypeptide comprises the amino acidsequence of a first FOXP polypeptide, wherein a zinc finger and leucinezipper region of the first FOXP polypeptide has been replaced with azinc finger and leucine zipper region of a second FOXP polypeptide. Insome embodiments, the first FOXP polypeptide is a FOXP1 polypeptide andthe second FOXP polypeptide is a FOXP2, FOXP3, or FOXP4 polypeptide. Insome embodiments, the first FOXP polypeptide is a FOXP2 polypeptide andthe second FOXP polypeptide is a FOXP1, FOXP3, or FOXP4 polypeptide. Insome embodiments, the first FOXP polypeptide is a FOXP3 polypeptide andthe second FOXP polypeptide is a FOXP1, FOXP2, or FOXP4 polypeptide. Insome embodiments, the first FOXP polypeptide is a FOXP4 polypeptide andthe second FOXP polypeptide is a FOXP1, FOXP2, or FOXP3 polypeptide. Insome embodiments, the first FOXP polypeptide is a FOXP3 polypeptide andthe second FOXP polypeptide is a FOXP1 polypeptide. In some embodiments,the first FOXP polypeptide is a FOXP1 polypeptide and the second FOXPpolypeptide is a FOXP3 polypeptide.

In some embodiments, a variant FOXP polypeptide comprises an amino acidsequence having at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 28. In someembodiments, a variant FOXP polypeptide comprises an amino acid sequenceof SEQ ID NO: 28.

Also embodied in the present disclosure are nucleic acid moleculescomprising a nucleotide coding sequence for the variant FOXPpolypeptides described herein. Given that the genetic code is well-knownin the art, it is routine for one of ordinary skill in the art togenerate such degenerate nucleic acid molecules that encode transgenes,including the variant FOXP polypeptides of the present disclosure. Forexample, an isolated nucleic acid molecule may comprise a nucleic acidsequence encoding any one of the variant FOXP polypeptides describedherein, including those referred to in the above paragraphs. In someembodiments, an isolated nucleic acid comprises the nucleotide sequenceof SEQ ID NO: 29.

In some embodiments, an isolated nucleic acid molecule comprises a NOX1core promoter or a variant NOX1 core promoter and a wildtype or variantFOXP3 polypeptide. In some embodiments, an isolated nucleic acidmolecule comprises a NOX1 core promoter or a variant NOX1 core promoterand IL10. In some embodiments, an isolated nucleic acid moleculecomprises a NOX1 core promoter or a variant NOX1 core promoter and awildtype or variant FOXP3 polypeptide and IL10. In some embodiments, anisolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:34.

Exemplary Vectors

As used herein, “vector” includes any genetic element, including, butnot limited to, a plasmid, phage, transposon, cosmid, chromosome,artificial chromosome, minichromosome, expression vector, virus, virion,etc., which is capable of replication when associated with the propercontrol elements and which can transfer nucleic acid molecules to cells.The term includes cloning and expression vectors, as well as viralvectors.

In some embodiments, one or more of the vectors, or all of the vectors,may be DNA vectors. In some embodiments, one or more of the vectors, orall of the vectors, may be RNA vectors. In some embodiments, one or moreof the vectors, or all of the vectors, may be circular. In otherembodiments, one or more of the vectors, or all of the vectors, may belinear. In some embodiments, one or more of the vectors, or all of thevectors, may be enclosed in a lipid nanoparticle, liposome, non-lipidnanoparticle, or viral capsid.

Non-limiting exemplary viral vectors include adeno-associated virus(AAV) vector, lentivirus vectors, adenovirus vectors, helper-dependentadenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors,bacteriophage T4, baculovirus vectors, pox virus vectors, and retrovirusvectors. In some embodiments, the viral vector may be an AAV vector. Inother embodiments, the viral vector may a lentivirus vector. In someembodiments, the lentivirus may be non-integrating. In some embodiments,the viral vector may be an adenovirus vector. In yet other embodiments,the viral vector may be an HSV-1 vector. In some embodiments, theHSV-1-based vector is helper dependent, and in other embodiments it ishelper independent. In additional embodiments, the viral vector may bebacteriophage T4. In further embodiments, the viral vector may be abaculovirus vector. In yet further embodiments, the viral vector may bea retrovirus vector.

An AAV vector may comprise a nucleic acid molecule enclosed in an AAVviral capsid. The encapsidated nucleic acid molecule may comprise AAVinverted terminal repeats (ITRs) positioned at each termini. AAV ITRsmay be derived from any number of AAV serotypes, including AAV2. AAVITRs can form hairpin structures and are involved in AAV proviralintegration and vector packaging. An AAV vector may comprise a capsidprotein from any one of the AAV serotypes, including, but not limited toAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, and variant capsids based on any serotype modified totarget a specific cell type. In some embodiments, an AAV vectorcomprises a nucleic acid molecule with AAV2 ITRs and enclosed in an AAV8viral capsid.

AAV vectors may be prepared using any one of the number of methodsavailable to those of ordinary skill in the art. Hermonat P L, et al.,1984b; Liu Y, et al., 2001; Grimm D, et al., 1998; Neyns B, et al.,2001; Cecchini S, et al., 2011.

In some embodiments, a vector comprises a nucleic acid moleculecomprising a nucleic acid molecule encoding a transgene that isoperatively linked to a promoter. The phrases “operatively positioned,”“operatively linked,” “under control,” or “under transcriptionalcontrol” means that a promoter is in the correct location andorientation in relation to the nucleic acid molecule to control RNApolymerase initiation and expression of the transgene.

In some embodiments, a vector may comprise one copy of a nucleotidesequence encoding a transgene. In other embodiments, the vector systemmay comprise more than one copy of a nucleotide sequence encoding atransgene. In some embodiments, a vector may comprise one or morenucleotide sequences encoding one or more transgenes. In someembodiments, a vector comprises a nucleic acid molecule encodingmultiple transgenes. In some embodiments, a vector comprises a nucleicacid molecule encoding two transgenes. In some embodiments, a vectorcomprises a nucleic acid molecule encoding three transgenes.

In some embodiments, the vector may be capable of driving expression ofone or more coding sequences, such as the coding sequence of a transgenedisclosed herein, in a host cell, either in vivo, ex vivo, or in vitro.In some embodiments, the host cell is a eukaryotic cell, such as, e.g.,a yeast, plant, insect, or mammalian cell. In some embodiments, the hostcell is a mammalian cell. In some embodiments, the host cell is a rodentcell. In some embodiments, the host cell is a human cell. In someembodiments, the host cell is a smooth muscle cell, a cardiac musclecell, a fibroblast cell, an immune cell (e.g., a macrophage, a T-cell,or a B-cell).

In some embodiments, a vector comprises a NOX1 core promoter or avariant NOX1 core promoter as disclosed herein. In some embodiments, avector comprises a nucleic acid molecule encoding a wildtype FOXPpolypeptide or variant FOXP3 polypeptide operably linked to a NOX1 corepromoter or a variant NOX1 core promoter. In some embodiments, a vectorcomprises a nucleic acid molecule encoding a variant FOXP3 polypeptideoperably linked to a promoter. In some embodiments, a vector comprises anucleic acid molecule encoding a variant FOXP3 polypeptide operablylinked to a regulated promoter. In some embodiments, a vector comprisesa nucleic acid molecule encoding a variant FOXP3 polypeptide operablylinked to a pathology-specific promoter. In some embodiments, a vectorcomprises a nucleic acid molecule encoding a variant FOXP3 polypeptideoperably linked to a NOX1 core promoter. In some embodiments, a vectorcomprises a nucleic acid molecule encoding a variant FOXP3 polypeptideoperably linked to a variant NOX1 core promoter.

In some embodiments, a vector comprises a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 3. In some embodiments,a vector comprises a nucleic acid molecule comprising at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3.In some embodiments, a vector comprises a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 4. In some embodiments,a vector comprises a nucleic acid molecule comprising at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4.

In some embodiments, a vector comprises a nucleic acid molecule encodinga variant FOXP3 polypeptide comprising the amino acid sequence of SEQ IDNO: 28. In some embodiments, a vector comprises a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 29. In someembodiments, a vector comprises a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 34.

In some embodiments, a vector comprises a nucleic acid molecule encodinga reporter protein operably linked to a NOX1 core promoter or a variantNOX1 core promoter. In some embodiments, a vector comprises a nucleicacid molecule encoding mCherry operably linked to a NOX1 core promoteror a variant NOX1 core promoter. In some embodiments, a vector comprisesa nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:22.

Exemplary Therapeutic Testing Model of Established and OngoingAtherosclerosis

An in vivo animal model of established and ongoing atherosclerosis fortesting treatment options that more closely mimics the human clinicalsituation is also described.

In some embodiments, a low density lipoprotein receptor knockout(LDLR-KO) or Apo E knockout mice or rat is fed a high cholesterol diet(HCD) for a period of time, such as at least 56 days, beforeadministering a therapeutic agent (e.g., a gene therapy vectorexpressing a therapeutic protein).

LDLR-KO mice fed a HCD develop vascular pathology, including immune cellarterial influx, smooth muscle cell proliferation, and atheroscleroticplaque formation. Getz G S and Reardon C A, 2005; Li D, et al., 2006;Liu Y, et al., 2005; Pan J H, et al., 2004; Chen J, et al., 2018.

A “high cholesterol diet” or “HCD” includes any natural or syntheticfat, such as lard, cocoa butter, cholate, etc. Exemplary HCDs arecommercially available (see., e.g.,criver.com/products-services/research-models-services/preconditioning-services/custom-diets?region=3611;dyets.com/experimental-diets/) and described in the literature (see,e.g., Zadelaar S, et al., 2007). In some embodiments, a HCD may compriseat least about 0.1%, at least 0.15%, at least 0.2%, at least 0.4%, atleast 0.5%, at least 1%, at least 1.25%, at least 1.5%, at least 2%, atleast 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, atleast about 5% cholesterol. In some embodiments, a HCD comprises between0.1% and 5% cholesterol. In some embodiments, a HCD comprises between 1and 15% cocoa butter. In some embodiments, a HCD comprises 4%cholesterol and 10% cocoa butter.

In some embodiments, a method for screening a therapeutic agentcomprises maintaining an LDLR-KO or Apo E knockout animal on a HCD andadministering a therapeutic agent to the animal no earlier than 56 daysafter beginning the HCD. In some embodiments, the therapeutic agent isadministered no earlier than 63 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 63 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 70 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 77 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 84 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 91 days after beginning the highcholesterol diet. In some embodiments, the therapeutic agent isadministered no earlier than 98 days after beginning the highcholesterol diet.

In some embodiments, the blood flow velocity, cross-sectional area ofthe aorta lumen, and/or the aortic wall thickness of the animal areassessed by ultrasound imaging. Exemplary methods for high ultrasoundimaging are described in Example 8.

Exemplary Pharmaceutical Compositions and Uses

“Pharmaceutical composition” refers to a preparation which is in suchform as to permit administration of a therapeutic agent and othercomponent(s) contained therein to a subject and does not containcomponents that are unacceptably toxic to a subject.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. A pharmaceutical composition may be in the form of solid,semisolid, liquid, cream, gel, capsule, or patch. The pharmaceuticalcomposition may be in a form that allows for slow release or delayedrelease of a therapeutic agent.

Examples of pharmaceutically acceptable carriers include alumina;aluminum stearate; lecithin; serum proteins, such as human serumalbumin, canine or other animal albumin; buffers such as phosphate,citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassiumsorbate; partial glyceride mixtures of saturated vegetable fatty acids;water; salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulose-based substances; polyethylene glycol; sucrose;mannitol; or amino acids including, but not limited to, arginine.

“Therapeutic agent,” as used herein, refers to an agent used for thetreatment or prevention of a disease, condition, or disorder. Atherapeutic agent may include a nucleic acid molecule, polypeptide,vector, and/or cell disclosed herein.

The pH of a pharmaceutical composition is typically in the range of fromabout pH 6 to pH 8 when administered, for example about 6, about 6.2,about 6.4, about 6.6, about 6.8, about 7, about 7.2. Pharmaceuticalcompositions may be sterilized if they are to be used for therapeuticpurposes. Sterility can be achieved by any of several means known in theart, including by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Sterility may be maintained with or withoutanti-bacterial agents.

In some embodiments, a nucleic acid molecule, polypeptide, vector, cell,and/or pharmaceutical composition disclosed herein can be utilized inaccordance with the methods herein to treat a vascular disease, acardiovascular disease, atherosclerosis, an inflammation-associateddisease, an age-associated disease, arthritis, and/or dementia.

In some embodiments, a nucleic acid molecule, a polypeptide, a vector, acell, and/or a pharmaceutical composition disclosed herein are used fortreating a subject having a vascular disease and/or a cardiovasculardisease. Exemplary forms of vascular or cardiovascular disease include,but are not limited to, general atherosclerosis; plaque formation;cholesterol accumulation; stenosis; restenosis; hypertension; peripheralartery disease; peripheral artery disease of the legs; peripheral arterydisease of the arms; peripheral artery disease of the gut/mesentery;disease of the carotid arteries; heart failure; cellular proliferationof smooth muscle; stroke; cardiac disease (e.g., risk stratification ofchest pain and interventional procedures); pulmonary circulatorydisease; graft occlusion or failure; need for or an adverse clinicaloutcome after peripheral bypass graft surgery; Paget-Schroetter disease;Budd-Chiari syndrome; peripheral vascular disease; renalatherosclerosis; renal vein thrombosis; jugular vein thrombosis;arterial disease of the aorta; carotid artery disease; coronary arterydisease; coronary heart disease; pulmonary circulatory disease;correction of adverse clinical outcome after surgery for coronary arterybypass (CABG); pulmonary embolism; ischemic diseases; multiplicity ofother cardiovascular disease related to obesity or an overweightcondition; thrombosis formation (e.g., venous thrombosis, deep veinthrombosis, portal vein thrombosis, cerebral venous sinus thrombosis, orarterial thrombosis) or other thrombotic events or complications;myocardial infarction; graft failure; vein graft failure; vein graftocclusion; autologous vein grafts; coronary revascularization; kidneyfailure; cerebrovascular disease; ischemia reperfusion injury; generalischemia; heart disease; cardiopulmonary resuscitation; myocardialinfection; treatment of adverse outcome after angioplasty; endothelialdysfunction; impaired general circulation; and left ventricularhypertrophy-acute coronary syndrome. A subject may have multiple formsof a vascular or cardiovascular disease.

In some embodiments, a nucleic acid molecule, a polypeptide, a vector, acell, and/or a pharmaceutical composition disclosed herein are used fortreating a subject having atherosclerosis.

In some embodiments, a nucleic acid molecule, a polypeptide, a vector, acell, and/or a pharmaceutical composition disclosed herein are used fortreating a subject having an inflammation-associated disease. In someembodiments, a nucleic acid molecule, a polypeptide, a vector, a cell,and/or a pharmaceutical composition disclosed herein are used fortreating a subject having an age-associated disease. In someembodiments, a nucleic acid molecule, a polypeptide, a vector, a cell,and/or a pharmaceutical composition disclosed herein are used fortreating a subject having arthritis, such as psoriatic arthritis,rheumatoid arthritis, or gouty arthritis. In some embodiments, a nucleicacid molecule, a polypeptide, a vector, a cell, and/or a pharmaceuticalcomposition disclosed herein are used for treating a subject havingasthma. In some embodiments, a nucleic acid molecule, a polypeptide, avector, a cell, and/or a pharmaceutical composition disclosed herein areused for treating a subject having macular degeneration of the retina.In some embodiments, a nucleic acid molecule, a polypeptide, a vector, acell, and/or a pharmaceutical composition disclosed herein are used fortreating a subject having diabetes mellitus.

In some embodiments, a nucleic acid molecule, a polypeptide, a vector, acell, and/or a pharmaceutical composition disclosed herein are used fortreating a subject having dementia. Exemplary forms of dementia include,but are not limited to, Alzheimer's disease, dementia with Lewy bodies,vascular dementia, and frontotemporal dementia. A subject may havemultiple forms of dementia, such as Alzheimer's disease and vasculardementia.

Methods for administering a nucleic acid molecule, a polypeptide, avector, a cell, and/or a pharmaceutical composition disclosed hereininclude, but are not limited to, administering a therapeuticallyeffective dose of a nucleic acid molecule, polypeptide, vector, cells,and/or pharmaceutical composition of the disclosure to a subject.

As used herein, “subject,” “individual,” and “patient,” are usedinterchangeably to refer to an individual organism, a vertebrate, amammal, or a human. In some embodiments, “subject” means any animal(mammal, human, or other) patient that can be afflicted with one or moreof the diseases, conditions, or disorders described herein and is inneed of treatment.

As used herein, “treat” and other forms of the term, including“treating,” “treated, and “treatment,” relate to an approach forobtaining beneficial or desired clinical results. Treatment covers anyadministration or application of a therapeutic agent for a disease,condition, or disorder in a subject, including a human subject. Forpurposes of this disclosure, beneficial or desired clinical resultsinclude, but are not limited to, any one or more of: alleviation of oneor more symptoms; diminishment of extent of a disease, condition, ordisorder; preventing or delaying spread of a disease, condition, ordisorder; preventing or delaying recurrence of disease, condition, ordisorder; amelioration of the state of the disease, condition, ordisorder; inhibiting or slowing the progression of a disease, condition,or disorder or arresting its development; and remission (whether partialor total) of a disease, condition, or disorder. Also, encompassed by“treatment” is a reduction of pathological consequence of aproliferative disease. The methods provided herein contemplate any oneor more of these aspects of treatment. In-line with the above, the termtreatment does not require one-hundred percent removal of all aspects ofa disease, condition, or disorder.

A “therapeutically effective amount” of a therapeutic agent may varyaccording to factors such as the type of disease to be treated, thedisease state, the severity and course of the disease, the type oftherapeutic purpose, any previous therapy, the clinical history, theresponse to prior treatment, the discretion of the attending physician,age, sex, and weight of the subject, and the ability of the therapeuticagent to elicit a desired response in the subject. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the therapeutic agent are outweighed by the therapeuticallybeneficial effects. A therapeutically effective amount may be deliveredin one or more administrations. A therapeutically effective amountrefers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, a therapeutically effective amount of atherapeutic agent of the disclosure can be provided. For example, atherapeutically effective amount of a polypeptide of the disclosure maybe administered at a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50,100, 500, or 1,000 mg/kg. In some embodiments, a therapeuticallyeffective amount of a vector of the disclosure may be administered at adose of 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ viral particles (e.g.,encapsidated genomes (eg)). In some embodiments, a therapeuticallyeffective amount of a vector of the disclosure may be administered at adose of 1×10¹⁰ to 1×10¹³ viral particles, 1×10¹⁰ to 1×10¹² viralparticles, or 1×10¹⁰ to 1×10¹¹ viral particles. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test bioassays or systems. For example, a therapeuticallyeffective amount can be estimated initially either in cell cultureassays or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in other subjects. Generally,the therapeutically effective amount is dependent of the desiredtherapeutic effect. For example, the therapeutically effective amount ofa vector of the disclosure can be assessed in a mouse model ofatherosclerosis.

A nucleic acid molecule, polypeptide, vector, cells, and/orpharmaceutical composition of the disclosure can be administered by anyone or more routes known in the art or described herein, for example,orally (e.g., in capsules, suspensions or tablets), parenterally (e.g.,intravenously or intramuscularly by solution, suspension, emulsion, orgel), intraperitoneal, rectal, cutaneous, nasal, vaginal, inhalant, skin(patch), and/or ocular. The nucleic acid molecule, polypeptide, vector,cells, and/or pharmaceutical composition of the disclosure may beadministered in any dose or dosing regimen. In some cases,administration may be accomplished by an ex vivo route. Ex vivo deliveryinvolves ex vivo (outside the body) transduction of host cells (e.g.,smooth muscle cells, cardiac muscle cells, fibroblast cells, immunecells such as macrophages, T-cells, and/or B-cells) by recombinantvectors, followed by administration of the transduced cells to thesubject. For example, host cells may be obtained from a subject,transduced outside the body by a vector of the disclosure, and thenadministered to the same subject or a different subject.

In some embodiments, a nucleic acid molecule, polypeptide, vector,cells, and/or pharmaceutical composition of the disclosure isadministered parenterally, by subcutaneous administration, intravenousinfusion, arterial injection, intramuscular injection, or injection intoa section of a ligated artery or vein, or a combination thereof.Ligation of an artery or vein may be accomplished by any methodunderstood by a person of ordinary skill in the art, such as by tying orclamping off a section of a vessel or artery (distal and proximal) so asto isolate the section from blood flow for a period of time. In someembodiments, a pharmaceutical composition in the form of a gel or creamand comprising the nucleic acid molecule, polypeptide, vector, and/orcells of the disclosure is applied to one or more locations of interest(e.g., an artery, vein, joint, etc.). In some embodiments, such a gel orcream may allow for slow release or delayed release of the therapeuticagent. In some embodiments, a nucleic acid molecule, polypeptide,vector, cells, and/or pharmaceutical composition of the disclosure isadministered as a bolus injection or by infusion over a period of time.

In some embodiments, a therapeutic agent is administered once in asingle dose or in multiple doses. When multiple doses are administered,the doses may be separated from one another by, for example, one hour,three hours, six hours, eight hours, one day, two days, one week, twoweeks, or one month. In some embodiments, a therapeutic agent isadministered every other day, once a week, or once a month. In someembodiments, a therapeutic agent is administered for, e.g., 2, 3, 4, 5,6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for anyparticular subject, specific dosage regimes should be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions. For example, the dosage of the therapeutic can beincreased if the lower dose does not provide sufficient therapeuticactivity.

In some embodiments, when a polypeptide encoded by a vector of thedisclosure is expressed in a subject, the expression level can beconstant over a desired period of time, for example, at least 1 week, atleast 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, atleast 3 months, at least 6 months, at least 1 year, or at least 5 years.In some embodiments, the expression of a polypeptide disclosed hereincan be sustained at or above a therapeutically effective dosage levelover a desired period of time.

In some embodiments, the amount of therapeutic agent is administered toa subject (in pulses, as continuous treatment or as a one-timeadministration (e.g., via gene therapy expression of a polypeptidedisclosed herein)) such that the blood levels of the polypeptide in thetreated subject are above about 20%, or above about 30%, or above about40%, or above about 50%, or between about 50-100% or above about 2-fold,or above about 3-fold, or above about 4-fold, or above about 5-fold ormore than 5-fold the blood levels of the endogenous polypeptide in acontrol subject.

EXAMPLES Example 1 Analysis of TGFβ and IL10 Expression in A7R5 RatSmooth Muscle Cells Expressing FOXP3

FOXP3 is a master transcription factor of the regulatory pathway in thedevelopment and function of regulatory T cells (Treg cells), which arespecialized T cells that act to suppress immune response. Loss of FOXP3expression results in increased chronic autoimmunity. Ziegler S, 2006.Expression of FOXP3 is generally understood to be restricted to Tregcells. Karagiannidis C, et al., 2004; Fontenot J D, et al., 2003; Hon S,et al., 2003; Khattri R, et al., 2003; Li Z, et al., 2015; Kitoh A, etal., 2009. Hence, downstream effects of FOXP3 expression in other celltypes, such as vascular smooth muscle cells, which are a target celltype in AAV treatment of atherosclerosis is unclear.

For example, expression levels of TGFβ and IL10 in immortal tissueculture A7R5 rat smooth muscle cells expressing FOXP3 was tested. TheA7R5 cells were co-transfected with pSV40-Neo and CMV-FoxP3 expressionplasmids at a 1:10 ratio and clones were selected with G418.G418-selected cell lines were tested for FOXP3 expression byQuantitative RT-PCR (Q-RT-PCR) for FOXP3 mRNA and compared to negativecontrols (untransfected A7R5 cells (labeled “A7R5”) and A7R5 cellstransfected with pSV40-Neo only and G418-selected (labeled “neo”)) (FIG.1A).

Relative expression of TGFβ and IL10 in select FOXP3-expressing cloneswas also measured by Q-RT-PCR for TGFβ and IL10 mRNA (FIG. 1B and FIG.1C, respectively) and compared to the same negative controls (“A7R5” and“neo”). While an increase in TGFβ expression was observed with someFOXP3-expressing clones, IL10 expression did not appear to be induced byFOXP3 expression.

Example 2 Exemplary Core NOX1 Promoter

Constitutive or systemic expression of anti-inflammatory proteins, suchas FOXP3, IL10, or TGFβ may not be desirable. For example, systemicallyexpressed or high circulating levels of IL10 has been associated withincreased bacterial and viral infections. Filippi C M and von Herrath MG, 2008; Zobel K, et al., 2012; Clemons K V, et al., 2000; Brooks D G,et al., 2008; Brooks D G, et al., 2006; Ejrnaes M, et al., 2006; Zeni E,et al., 2007; Maris C H, et al., 2007; Asadullah K, et al., 2003. Inaddition, many promoters used in gene therapy, including regulatablepromoters, are problematic due to their large size and the limitedpackaging capacity of AAV. For example, the LOX1 high-lipid regulatablepromoter (˜2,400 bp) is too large for regulating multiple gene productsin the context of AAV. When the LOX1 promoter is used, only 2 kb of AAVpackaging capacity remains available for transgenes.

Expression of NOX1 is understood to be upregulated in response to shearstress, high lipids, and other irritating challenging agents, such asinterferon gamma (IFN-γ) and Angiotensin-2 (Ang II). Hwang J, et al.,2003; Hsieh J H, et al., 2014; Valente A J, et al., 2007; Kuwano Y, etal., 2005; Nguyen Dinh Cat A, et al., 2012; Manea A., et al., 2009.

An exemplary NOX1 promoter having enhanced features (SEQ ID NO: 4;“eNOX1 promoter”) comprising a variant NOX1 core promoter (SEQ ID NO: 3)with three NFκB binding sequences (ggggattccc; SEQ ID NO: 19), and twoOct1 binding sequences (atgcaaat; SEQ ID NO: 21) added to the 5′ end wasdeveloped. The variant NOX1 core promoter sequence (SEQ ID NO: 3) of theeNOX1 promoter includes nucleotides 1562-2027 of the NOX1 gene (GenBankAccession No. DQ314883.1) (SEQ ID NO: 2) with two base changes (thefirst to enhance the CAAT box and the second to knock out an unnecessaryATG start site) and a short downstream sequence (including a TGA stopsite and Kozak consensus sequence). Unlike the LOX1 promoter, the smallsize (483 bp) of the eNOX1 promoter allows for the inclusion of multipletherapeutic genes in the context of an AAV vector.

Furthermore, inclusion of transcription factor binding sequences, suchas one or more NFκB and/or Oct1 binding sequences, may improveexpression in response to certain stimuli. For example, NF-κB is atranscription factor activated in cells during disease progression andin response to cellular stress. Ganguli A, et al., 2005; Mercurio F andManning A M, 1999; Zhang Q, et al., 2017. In the context of LOX-1promoter studies, the NFκB binding motif has been associated with AngII-induced promoter activation and the Oct-1 binding motif has beenassociated with Ox-LDL-induced promoter activation. Chen J, et al., BCJ,2006; Chen J, et al., ATVB, 2006. However, Oct-1 has also been shown torepress NFκB-dependent gene expression (Dela Paz N G, et al., 2006;Voleti B and Agrawal A, 2005), leading to uncertainty as to whether aNOX1 core promoter with additional NFκB and Oct1 binding sequences couldactivate gene expression in the presence of disease-associated stimuli.

The ability of the eNOX1 promoter (SEQ ID NO: 4) to regulate expressionof FLAG-mCherry in vitro in the presence of factors associated withinflammation, specifically Angiotensin II (Ang II), Lipopolysaccharides(LPS), and Oxidized Low Density Lipoprotein (Ox-LDL) was tested. Ang IIis a peptide hormone that causes vasoconstriction and increased bloodpressure. LPS stimulates the release of inflammatory cytokines leadingto acute inflammatory response and, in extreme cases, anaphylacticshock. Ox-LDL is a major driver of vascular pathology in humans.

HEK 293 cells were transfected with 1 μg of a plasmid comprising theeNOXpr-3xFLAG-mCherry construct (SEQ ID NO: 22) and cultured inDulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10%(v/v) fetal bovine serum (FBS), 10 U/mL penicillin. The cell cultureswere treated with increasing concentrations of Ang II (Sigma, USA) (0ng/ml, 100 ng/ml, 1 μg/ml, and 10 μg/ml), LPS (Sigma, USA) (0 ng/ml, 10ng/ml, 100 ng/ml, and 1 μg/ml), or Ox-LDL (Yiyuanbiotech, China) (0ng/ml, 10 ng/ml, 1 μg/ml, and 60 μg/ml) at 36 hours and harvested at 60hours.

Cell extracts were prepared and separated by SDS-PAGE. Western blotanalysis was performed using mouse anti-FLAG primary antibody (diluted1:1000, F-1804, Sigma, USA), mouse anti-β-actin primary antibody(diluted 1:8000, A1978, Sigma, USA) as a loading control, and secondarymouse anti-IgG ALEXA Flour 647 (diluted 1:500, Invitrogen, USA). Therelative expression of FLAG and β-actin was visualized and quantifiedusing the enhanced chemiluminescence light method using NIH ImageJsoftware. Results are shown as the mean±SD from three independentexperiments.

FIG. 2 shows relative expression levels of FLAG-mCherry under thecontrol of the eNOX1 promoter in the presence of increasingconcentrations of Ang II (FIG. 2A), LPS (FIG. 2B), and Ox-LDL (FIG. 2C)compared to an untransfected control. The expression of FLAG-mCherryunder control of the eNOX1 promoter trended to increase with theconcentration of Ang II, LPS, and ox-LDL, and at the highestconcentration of the stimulant, expression was statistically higher thanthe 0 μg/ml controls.

The relative expression of FLAG-mCherry was also assessed byfluorescence microscopy. Similar to the prior study, HEK 293 cells weretransfected with 1 μg of the eNOXpr-3xFLAG-mCherry plasmid or aCMVpr-FLAG-mCherry plasmid. Cells transfected with eNOXpr-3xFLAG-mCherrywere treated with increasing concentrations of Ang II (0 ng/ml, 100ng/ml, 1 μg/ml, and 10 μg/ml), LPS (0 ng/ml, 10 ng/ml, 100 ng/ml, and 1μg/ml), or Ox-LDL (0 ng/ml, 100 ng/ml, 1 μg/ml, and 60 μg/ml) at 36hours and harvested at 60 hours. Relative expression of FLAG-mCherry wasvisualized and quantified by fluorescence microscopy (Lica, USA) andImageJ software.

FIG. 3 shows relative expression levels of FLAG-mCherry under thecontrol of the eNOX1 promoter in the presence of increasingconcentrations of Ang II (FIG. 3B), LPS (FIG. 3C), and Ox-LDL (FIG. 3D).Data are expressed as mean±SD (*P<0.05). Expression of mCherry under theconstitutive control of the CMV promoter and an untransfected controlare shown in FIG. 3A. The expression of FLAG-mCherry under control ofthe eNOX1 promoter trended to increase with the concentration of Ang II,LPS, and Ox-LDL, and at the highest concentration expressions werestatistically higher than the 0 μg/ml controls.

Example 3 In Vivo AAV Expression with an Exemplary NOX1 Core Promoter

The responsiveness of the eNOX1 promoter (SEQ ID NO: 4) was tested inthe context of an AAV2/8 expression vector in a high lipid mouse model.Low density lipoprotein receptor knockout (LDLR-KO) mice fed a highcholesterol diet (HCD) develop vascular pathology, including immune cellarterial influx, smooth muscle cell proliferation, and atheroscleroticplaque formation. Getz G S and Reardon C A, 2005; Li D, et al., 2006;Liu Y, et al., 2005; Pan J H, et al., 2004; Chen J, et al., 2018. Thelevel of expression from the eNOX1 promoter was monitored over time asthe vascular pathology of the LDLR-KO mice progressed. The expressionconstruct tested included a FLAG-mCherry fusion protein under thecontrol of the eNOX1 promoter with flanking AAV2 inverted terminalrepeats (ITRs) and was packaged into AAV serotype 8 capsid under GMPconditions by OBiO Technology Corp., Ltd. (Shanghai, China). Theexperimental design for testing AAV2/8.eNOXpr-3xFLAG-mCherry is shown inFIG. 4A.

The LDLR-KO strain (B6; 12957-LdlrtmlHer/J) was purchased from JacksonLaboratories (Bar Harbor, Me., USA). The mice were group-housed underconstant temperature (23±2° C.) and 40-60% humidity with humane care andmaintained on a 12 hour/12 hour, light/dark cycle. Food and water wereaccessed ad libitum. Study protocols about laboratory animal use were inaccordance with the guidelines for the Beijing Friendship HospitalAnimal Care and Ethics Committee.

Three groups of eight-week old LDLR-KO mice weighing 16-20 grams wereused in the 20-week experiment. Group 1 (n=12) was a negative controlgroup of untreated LDLR-KO mice maintained on a normal diet (ND) fromday 0 to week 6 (n=5), from day 0 to week 12 (n=4), or from day 0 toweek 20 (n=3). Groups 2 and 3 received AAV2/8.eNOXpr-3xFLAG-mCherry onday 0 at a titer of 1×10¹¹ viral encapsidated genomes (eg)/mL via tailvein injection of 200 uL virus per mouse, followed by two boosterinjections at an interval of approximately 2 days. Group 2 (n=14) was asecond negative control group and received the ND from day 0 to week 6(n=4), day 0 to week 12 (n=4), or day 0 to week 20 (n=6). Group 3 (n=15)received a high cholesterol diet (HCD) from day 0 to week 6 (n=4), fromday 0 to week 12 (n=4), or from day 0 to week 20 (n=7). The HCD, whichconsisted of 4% cholesterol and 10% Coco butter (Beijing HFK BioscienceCompany, China), was used to ensure the development of atherosclerosis.At weeks 6, 12, and 20, animals were humanely sacrificed, and tissuesharvested for eNOX1 promoter expression analysis.

Regulated expression of FLAG-mCherry under the eNOX1 promoter in liverat 6 weeks, 12 weeks, and 20 weeks was measured by immuno-fluorescencestaining for FLAG (FIG. 5B). Frozen sections were cryostat-microtomeshaved from frozen liver tissues. Slides of the tissues were incubatedwith primary antibodies against FLAG (dilution 1:100, F-1804, Sigma,USA) overnight at 4° C. After washing with phosphate-buffered saline(PBS) for three times, the tissues were incubated with a mixture ofAlexa Fluor 647 anti-mouse secondary antibody (dilution 1:500,Invitrogen, USA) at room temperature for 1 hour. The sections werecounter-stained with DAPI. The stained tissue was viewed andphotographed using a confocal microscope (Olympus, Japan) at 20× and 60×magnification.

The activity of the eNOX1 promoter is shown at the 6, 12, and 20 weektime points in representative sections in FIG. 5B. Immunofluorescencestaining shows that following injection of AAV2/8.eNOXpr-3xFLAG-mCherry,more transgene expression is observed among the HCD-fed mice than theND-fed mice at each of the three time points. FLAG-mCherry expressioncontinued to rise in the HCD-fed group from week 6, through week 12,with the highest level of expression observed at week 20. In addition,blood vessel and liver hepatocyte cell transduction were observed.

In vivo expression of AAV2/8.eNOXpr-3xFLAG-mCherry was compared betweenND-fed mice and HCD-fed mice at the 20-week time point by Western blotanalysis using protein extracted from frozen liver tissues (FIG. 5C).Western blot analysis was performed using mouse anti-FLAG primaryantibody (diluted 1:1000, F-1804, Sigma, USA) and mouse anti-β-actinprimary antibody (diluted 1:8000, A1978, Sigma, USA) as a loadingcontrol. The membranes were visualized by the enhanced chemiluminescencelight method. Densitometric results using ImageJ software show thatexpression from the eNOX1 promoter was more than five-fold higher in theHCD-fed mice compared to the ND-fed mice at the 20-week time point (FIG.5E). Data are expressed as mean±SD (*P<0.05).

In vivo expression of AAV2/8.eNOXpr-3xFLAG-mCherry was also comparedbetween ND-fed mice at the 6-week time point and HCD-fed mice at the 6-,12-, and 20-week time points by Western blot analysis using proteinextracted from frozen liver tissues (FIG. 5D). Western blot analysis wasperformed as described in the above paragraph. Densitometric resultsshow that expression from the eNOX1 promoter increased in atime-dependent manner in the HCD-fed mice and was more than four-foldhigher in the HCD-fed mice at 20-weeks compared to the ND-fed controlmice (FIG. 5F). Data are expressed as mean±SD (*P<0.05).

Collectively, the immunofluorescence and Western analyses are consistentwith the Ox-LDL-stimulated eNOX1 promoter expression observed in HEK 293cells (FIG. 2 and FIG. 3). FLAG-mCherry expression was observed in bloodvessels and hepatocytes of the liver. Further, as vascular pathology ofLDLR-KO mice fed a HCD diet progressed, the level of transcription fromthe eNOX1 promoter of the AAV2/8.eNOXpr-3xFLAG-mCherry was shown to alsotemporally increase. These data suggest that therapeutic expression fromthe eNOX1 promoter increases in the face of increasing vascularpathology occurring over time (see FIG. 5A).

Example 4 Modification of FOXP3 Zinc Finger and Leucine Zipper Region

Forkhead box proteins (FOXP) are a family of transcription factors(e.g., FOXP1, FOXP2, FOXP3, and FOXP4) involved in regulating theexpression of genes involved in development, immune disorders, andcancer progression. FOXP family proteins have a zinc finger and leucinezipper region understood to be involved in protein dimerization. Kim J,et al., 2019; Wang B, et al., 2003; Song X, et al., 2012. Presumably, aFOXP expressed in vivo from an AAV vector could dimerize with itsendogenous FOXP counterpart. To enhance homodimerization of FOXPexpressed from an AAV vector and increase the likelihood that efficacyis due to the AAV-delivered FOXP, replacement of one FOXP zinc fingerand leucine zipper region with that of another FOXP was explored.

A chimeric FOXP3/FOXP1 (“FOXP3(P1)”) protein was designed in which anamino acid sequence from the zinc finger and leucine zipper region ofhuman FOXP3 was replaced with an analogous amino acid sequence from thezinc finger and leucine zipper region of FOXP1. The FOXP3(P1) amino acidsequence (SEQ ID NO: 28), which is 431 amino acids in length, includesamino acids 1 to 225 and 264 to 431 of human FOXP3 (NCBI ReferenceSequence NP_054728.2; SEQ ID NO: 24) flanking amino acids 147 to 184 ofhuman FOXP1 (GenBank: AF146698.2; SEQ ID NO: 26). The FOXP3 and FOXP1zinc finger and leucine zipper regions that were interchanged share onlyabout 45% homology. The resulting FOXP3(P1) chimeric protein (SEQ ID NO:28) should preferentially homodimerize over dimerizing with endogenousFOXP3. Potential heterodimerization of FOXP3(P1) with endogenous FOXP1is unlikely to be harmful since endogenous FOXP3 and FOXP1 areunderstood to already form heterodimers. Ren J, et al., 2019; KonopackiC, et al., 2019; Li B, et al., 2007; Rudra D, et al, 2012; Deng G, etal, 2019.

Example 5 FOXP3(P1) and IL10 Dual Gene AAV Vector

Vascular smooth muscle cells are a target cell type in the AAV treatmentof atherosclerosis. Based on the studies described in Example 1, anincrease in TGFβ expression but not IL10 expression was observed inFOXP3-expressing smooth muscle cells. Inclusion of nucleotide sequencesfor both FOXP3(P1) and IL10 in an AAV vector may increase thetherapeutic efficacy observed, such as regression of vascularpathologies and/or prevention of further development of vascularpathologies. To reduce the overall expression of FOXP3(P1) and IL10, useof a small lipid-responsive promoter was explored. The eNOX1 promoterdescribed in Example 2 allows for the inclusion of multiple therapeuticgenes in the context of an AAV vector, including both FOXP3(P1) andIL10.

Design of a three-component expression construct(eNOX1pr-FoxP3(P1)-IL10; SEQ ID NO: 34) totaling 2675 nucleotidescomprising the eNOX1 promoter (SEQ ID NO: 4), FOXP3(P1) coding sequence(SEQ ID NO: 29), and a human IL10 coding sequence (SEQ ID NO: 32) isshown in FIG. 6. Since overexpression of IL10 can be detrimental, theconstruct was designed such that the IL10 coding sequence does not havea dedicated enhancer-promoter region. Instead, a synthetic TATA boxsequence was placed downstream of FOXP3(P1) and upstream of IL10allowing for limited expression from mRNA translation from the IL10coding sequence start methionine. A synthetic poly-adenylation sequencewas placed downstream of the IL10 coding sequence. The expressionconstruct with flanking AAV2 inverted terminal repeats (ITRs) waspackaged into AAV serotype 8 capsid (AAV2/8.eNOXpr-FoxP3(P1)-IL10) underGMP conditions by OBiO Technology Corp., Ltd. (Shanghai, China) for genedelivery into mice.

Example 6 Administration of AAV2/8.eNOXpr-FoxP3(P1)-IL10 to an AnimalModel of Established and Ongoing Atherosclerosis

Therapeutic benefit of dual expression of FOXP3 (P1) and IL10 fromeNOX1pr-FoxP3(P1)-IL10 gene delivery against established and ongoingatherosclerotic disease was tested. The LDLR-KO model fed HCD developsvascular pathology, including immune cell arterial influx, smooth musclecell proliferation, and atherosclerotic plaque formation. In previousgene therapy treatment trials involving the LDLR-KO model, treatment bytail vein injection of the putative therapeutic AAV vector was initiatedat the same time as the HCD was initiated. Cao M, et al., 2015; Zhu H,et al., JTM 2014; Zhu H, et al., Plos One 2014; Cao M, et al., 2011;Khan J A, et al., 2011; Khan J A, et al., 2010; Dandapat A, et al.,BBRC, 2007; Dandapat A, et al., Gene Ther, 2007; Liu Y, et al., 2005.Hence, testing of a vector's therapeutic effect when present from day 0through week 20 may be more accurately described as a prevention modelas opposed to a treatment model.

This previous mouse model design of administering therapy at the sametime as initiating the HCD is not representative of the clinical settingwhere human patients are first diagnosed and then treated. Patientstypically seek medical intervention when they are symptomatic, and arelikely to have significant and established vascular pathology. An animaltreatment model that more closely mimics this clinical situation wasdeveloped in which mice are maintained on a HCD for several months priorto the administration of the therapeutic vector (e.g., as shown in FIG.7).

AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector was tested using the experimentaldesign shown in FIG. 4B. This therapeutic-focused study differed fromthe animal expression study of Example 3 and FIG. 4A by allowing for a12-week HCD-induced pathology accumulation period before therapeuticintervention. Three groups of eight-week old LDLR-KO mice were used in a20-week study. Group 1 was a negative control group of untreated LDLR-KOmice maintained on a normal diet (ND) from day 0 to week 20 (n=3).Groups 2 and 3 each received the HCD diet from day 0 through week 20.However, the AAV vector injection was delayed until week 12 to allow forHCD-induced pathology to accumulate. At week 12, Group 2 received theAAV2/8.eNOXpr-3xFLAG-mCherry vector (disease positive control) (n=9) andGroup 3 received the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector (experimentalgroup) (n=9) at a titer of 1×10¹¹ viral encapsidated genomes (eg)/mL viatail vein injection of 200 uL virus per mouse, followed by two boosterinjections at an interval of approximately 2 days. The high cholesteroldiet (HCD) comprised 4% cholesterol and 10% Coco butter. The animalswere assessed for therapeutic effects against HCD-inducedatherosclerosis by high resolution ultrasound using VisualSonicsVevo2100 at 20 weeks. Thereafter, the mice were euthanized by overdoseof 1% pentobarbital, and tissues harvested for other analyses.

The relative expression of FOXP3(P1) in liver (FIG. 8A) and heart (FIG.8B) and of IL10 in liver (FIG. 8C) and heart (FIG. 8D) in Group 2 andGroup 3 animals receiving AAV2/8.eNOXpr-3xFLAG-mCherry andAAV2/8.eNOXpr-FOXP3(P1)-IL10, respectively was determined at week 20 (8weeks after vector administration). Relative expression of the FOX3(P1)and IL10 mRNA was determined by quantitative, reverse transcription PCR(qRT-PCR). Total mRNA was extracted from liver and heart tissue usingQiagen RNeasy Mini Kit (Qiagen, Germany) and cDNA was reverselytranscribed. The qRT-PCR system and data analysis were performed inaccordance with Yang A T, et al. The primers used are listed in Table 4,below. The forward FoxP3(P1) primer is specific for FoxP3 and thereverse primer is specific for the FoxP1 leucine zipper/zinc fingerregion. FoxP3(P1) and IL10 expression was normalized to expression ofGAPDH.

TABLE 4 Primers for real-time PCR Gene Primers FoxP3(P1)Forward 5′-AAAGATAGTACGTTGTCCGCAG-3′ (SEQ ID NO: 37)Reverse 5′-ATTTGCACCCTGCATTGCGC-3′ (SEQ ID NO: 38) IL10Forward 5′-TCTGTGCTGCCTCGTGCTCC-3′ (SEQ ID NO: 39)Reverse 5′-TCTGACAGAGCCTGGCACCC-3′ (SEQ ID NO: 40) mGAPDHForward 5′-TCCACTCACGGCAAATTCAAC-3′ (SEQ ID NO: 41)Reverse 5′-CGCTCCTGGAAGATGGTGATG-3′ (SEQ ID NO: 42)

As shown in FIGS. 8A, 8B, 8C, and 8D, the presence of FoxP3(P1) and IL10mRNA was observed in liver and heart tissue of mice injected withAAV2/8.eNOXpr-FOXP3(P1)-IL10 (Group 3), but not in mice treated withAAV2/8.eNOXpr-3xFLAG-mCherry (Group 2). All data were expressed asmeans±SD (*P<0.05).

Total plasma cholesterol (TC, FIG. 9A), triglycerides (TG, FIG. 9B),low-density lipoproteins (LDL, FIG. 9C), and high-density lipoproteins(HDL, FIG. 9D) were measured at 20 weeks for each of Groups 1-3 miceusing an automated chemistry analyzer (AU480, Olympus, Japan). As shownin FIG. 9, blood lipid levels were high in both groups on HCD (Groups 2and 3) compared to the ND control group (Group 1). TheAAV2/8.eNOXpr-3xFLAG-mCherry (Group 2) and AAV2/8.eNOXpr-FOXP3(P1)-IL10(Group 3) animal groups were statistically different from the ND control(Group 1) with respect to TC, LDL, and HDL. All data were expressed asmeans±SD (*P<0.05).

Animal weight was measured by digital scale. Liver enzyme and albuminlevels were determined by automatic chemistry analyzer (AU480, Olympus,Japan) as a measure of potential immunological response to thetransgenes and AAV capsid proteins. FIG. 10 shows that animal weight(FIG. 10A) and levels of alanine aminotransferase (ALT, FIG. 10B),aspartate aminotransferase (AST, FIG. 10C), alkaline phosphatase (ALP,FIG. 10D), and albumin (ALB, FIG. 10E) were statistically similar amongall three animal groups suggesting no significant liver damage and nosignificant immune response to the transgenes or AAV8 capsid proteins atweek 20 (8 weeks post-vector administration).

Example 7 Histological and Staining Analysis Following Treatment withAAV2/8.eNOXpr-FoxP3(P1)-IL10

Effects of AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector compared to that ofAAV2/8.eNOXpr-3xFLAGmCherry on the aorta challenged with a highcholesterol diet were considered. Entire aortas from the animals in eachof Groups 1-3, including the aortic arches and the thoracic andabdominal regions, were removed for further analysis followingeuthanization of the animals by overdose with 1% pentobarbital. Theaortas were flushed with saline solution.

Whole aortas were fixed in 10% buffered formalin before Oil Red Ostaining of triglycerides, lipids, and lipoproteins to identifyatherosclerotic lesions, as previously described. Dandapat A, et al.,Gene Therapy, 2007; Li D, et al., 2006. Unstained small animal aortasnormally appear translucent, but show lipid deposition as white areas.The stained aortas were inspected under a dissecting microscope and anysmall pieces of adventitial fat that remained attached were removed verycarefully without disturbing the aorta itself as well as the internallipid accumulations (plaques). The stained aortas were photographedunder natural light using a 10-megapixel digital camera (Nikon, Japan).Images of representative aortas stained with Oil Red O from each animalgroup are shown in FIG. 11A and enlargements of the aortic arch areshown in FIG. 11B. More numerous areas of red color appear in both theaortic arch (FIG. 11B) and in the descending aorta (FIG. 11A) ofAAV2/8.eNOXpr-3xFLAGmCherry vector-treated mice fed a HCD (Group 2)compared to control mice receiving a normal diet and no vector (Group 1)or AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated mice fed a HCD (Group 3).

For histological staining, aortas were fixed in 4% paraformaldehyde,dehydrated using sucrose, and embedded in OCT (Sakura, USA).Representative histologic sections from each of the three animal groupswere Oil Red O/hematoxylin and eosin (H&E) stained (FIG. 11C) as well assimple H&E stained (FIG. 11D). The AAV2/8.eNOXpr-3xFLAGmCherryvector-treated group fed a HCD (Group 2) showed much higher levels ofred staining indicating more atherosclerotic plaque formation thaneither the normal diet control mice (Group 1) or theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated mice fed a HCD (Group 3),suggesting a therapeutic effect from FOXP3(P1)-IL10 gene delivery.Simple H&E stained sections showed similar results.

Example 8 High Resolution Ultrasound Analysis Following Administrationof AAV2/8.eNOXpr-FoxP3(P1)-IL10 to an Animal Model of Established andOngoing Atherosclerosis

Blood velocity is used as a general indicator of vascular health. Forexample, high blood velocity can be indicative of disease resulting fromvascular pathologies, such as atherosclerotic plaques. High resolutionultrasound (HRUS) imaging was used to measure blood velocity and aortawall thickness, as a measurement of therapeutic effect between the threeanimal groups at week 20: the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector-treated experimental group (Group 3), the ND negative controlgroup (Group 1), and the AAV2/8.eNOXpr-3xFLAGmCherry disease positivecontrol group (Group 2).

The animals were fasted for 8 hours before ultrasound imaging using aFujifilm VisualSonic Vevo2100 (Toronto, Canada) high-resolution imagingsystem with an RMV 70B transducer and having a center frequency of 30MHz. The mice were anesthetized using 1.5% isoflurane (Isothesia, AbbottLaboratories, Chicago, USA) with oxygen. The abdominal and thoracicregion hair was removed by chemical hair remover (Veet, USA). The micewere laid supine out on a thermostatically heated platform. A pre-warmedtransducing gel (Medline Industries, Inc., Mundelein, USA) was spreadover the skin as a coupling medium for more accurate measurements. Imageacquisition was started on B-mode and images of aortic arch, thoracic,and abdominal region of aorta were recorded. Then, aorta wall thicknessof aorta arch, thoracic and abdominal region of aorta were scanned onM-mode. Next, the scan head probe was turned 90° for a short-axis viewto visualize the cross-sectional area of the aorta. Measurement of theflow velocity, orientation of the abdominal aorta by ultrasound, wasaccomplished by tilting the platform and the head of mouse down with thetransducer probe towards the feet and tail of the mouse. Thispositioning resulted in the Doppler angle to be less than 60° foraccurate measurements of blood flow velocity in the pulse-wave Doppler(PW) mode within aorta arch and abdominal aorta. Off-line measurementsand data analysis were performed using the Vevo2100 Analytical Software.The complete imaging for each mouse lasted for about 25-30 min.

Blood flow velocities in the luminal center of the aorta were measuredby HRUS and representative images from the analysis were captured (FIG.12). The systolic blood velocity (systolic pulse wave velocity) in theaortic arch of the AAV2/8.eNOXpr-3xFLAGmCherry animals maintained on aHCD (Group 2; 676±94 mm/sec) was significantly higher (p<0.05) than thenegative control animals maintained on a ND (Group 1; 532±133 mm/sec),indicating the presence of vascular pathology in the disease group.Moreover, the aortic arch systolic blood velocity of theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated experimental animalsmaintained on a HCD (Group 3; 549±73 mm/sec) was about 70% lower(p<0.05) than the disease positive control group (Group 2) and nearlyreached the blood velocity level of the ND negative control group (Group1). All data were given as means±SD (*P<0.05).

FIG. 13 shows that the systolic blood velocity (systolic pulse wavevelocity) in the abdominal region of the aorta among animal groups. Theabdominal aortic systolic blood velocity of theAAV2/8.eNOXpr-3xFLAGmCherry animals maintained on a HCD (Group 2;681±129 mm/sec) was significantly higher than the negative controlanimals maintained on a ND (Group 1; 418±88 mm/sec), indicative ofvascular pathology in the disease group. Moreover, the abdominal aorticsystolic blood velocity of the AAV2/8.eNOXpr-FOXP3(P1)-IL10vector-treated experimental animals maintained on a HCD (Group 3; 495±78mm/sec) was about 63% lower (p<0.05) than the disease positive controlgroup (Group 2) and nearly reached the blood velocity level of the NDnegative control group (Group 1). All data were given as means±SD(*P<0.05).

HRUS was used to image and measure the cross-sectional area of thethoracic region of the aortas of each animal group (FIG. 14). Thecross-sectional area of the lumens of the aortas was significantlysmaller in the AAV2/8.eNOXpr-3xFLAGmCherry disease positive controlgroup (Group 2; 0.84±0.11 mm²) compared to the ND negative control group(Group 1; 1.47±0.22 mm²), which is consistent with the higher systolicblood velocity observed in the disease group. In addition, thecross-sectional area of the lumens of the aortas was significantlylarger in the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated experimentalgroup (Group 3; 1.00±0.17 mm²) than the disease positive control group(Group 2). That observation is also consistent with the lower bloodvelocity observed in the AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treatedgroup. All data were given as means±SD (*P<0.05).

HRUS was used to image and measure the wall thickness of the aorta archregion (FIG. 15). The images included in FIG. 15 are representative ofthose captured during the analysis. The aortic wall thickness of theAAV2/8.eNOXpr-3xFLAGmCherry disease positive control group (Group 2;0.54±0.88 mm) was significantly thicker than the ND negative controlgroup (Group 1; 0.21±0.02 mm), consistent with the presence of vascularpathology in the disease group. Notably, the aortic wall thickness ofthe AAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated experimental group(Group 3; 0.38±0.10 mm) was significantly thinner than the diseasepositive control group (Group 2). All data were given as means±SD(*P<0.05).

The HRUS analysis for systolic blood velocity, lumen cross sectionalarea, and wall thickness showed significantly less vascular pathologyfor the FOXP3(P1)-IL10 AAV vector-treated animals maintained on a HCD(Group 3) compared to the animals maintained on a HCD diet andadministered the FLAG-mCherry AAV vector (Group 2). The lower systolicblood velocities (FIG. 12 and FIG. 13), larger aortic lumens (FIG. 14),and thinner aortic wall thickness (FIG. 15) observed among theAAV2/8.eNOXpr-FOXP3(P1)-IL10 vector-treated animals maintained on a HCD(Group 3) evidences significantly less overall vascular pathologycompared to the AAV2/8.eNOXpr-3xFLAGmCherry animals maintained on a HCD(Group 2). These results are consistent with histological studiesdescribed in Example 7. Overall, these data suggest that FOXP3(P1)-IL10gene delivery was therapeutically efficacious in a mouse model ofestablished and ongoing atherosclerosis.

CITED REFERENCES

The complete disclosures of all publications cited herein areincorporated herein by reference in their entireties as if each wereindividually set forth in full herein and incorporated.

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EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. The foregoingdescription and Examples detail certain embodiments and describes thebest mode contemplated by the inventors. It will be appreciated,however, that no matter how detailed the foregoing may appear in text,the embodiment may be practiced in many ways and should be construed inaccordance with the appended claims and any equivalents thereof.

As used herein, the term about refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term about generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited range) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). When terms such as at leastand about precede a list of numerical values or ranges, the terms modifyall of the values or ranges provided in the list. In some instances, theterm about may include numerical values that are rounded to the nearestsignificant figure.

What is claimed is:
 1. An isolated variant FOXP polypeptide comprising afull-length wildtype FOXP3 polypeptide, except that a zinc finger andleucine zipper region of the wildtype FOXP3 polypeptide has beenreplaced with a zinc finger and leucine zipper region of a wildtypeFOXP1 polypeptide.
 2. The isolated variant FOXP polypeptide of claim 1,wherein the wildtype FOXP3 polypeptide comprises a human sequence andthe zinc finger and leucine zipper region of the wildtype FOXP1polypeptide comprises a human sequence.
 3. The isolated variant FOXPpolypeptide of claim 1, wherein the full-length wildtype FOXP3polypeptide comprises an amino acid sequence of SEQ ID NO: 24 and thezinc finger and leucine zipper region of the wildtype FOXP1 polypeptidecomprises the amino acid sequence of SEQ ID NO:
 27. 4. An isolatedvariant FOXP3 polypeptide comprising an amino acid sequence having atleast 97% sequence identity to the amino acid sequence of SEQ ID NO: 28.5. An isolated variant FOXP3 polypeptide comprising the amino acidsequence of SEQ ID NO:
 28. 6. An isolated nucleic acid comprising anucleic acid sequence encoding a variant FOXP polypeptide comprising afull-length wildtype FOXP3 polypeptide, except that a zinc finger andleucine zipper region of the wildtype FOXP3 polypeptide has beenreplaced with a zinc finger and leucine zipper region of a wildtypeFOXP1 polypeptide.
 7. The isolated nucleic acid of claim 6, wherein thevariant FOXP polypeptide comprises an amino acid sequence having atleast 97% sequence identity to the amino acid sequence of SEQ ID NO: 28.8. The isolated nucleic acid of claim 6, wherein the variant FOXPpolypeptide comprises the amino acid sequence of SEQ ID NO:
 28. 9. Anisolated contiguous nucleic acid comprising a first nucleic acidencoding a first therapeutic polypeptide and a second nucleic acidencoding a second therapeutic polypeptide, wherein the first nucleicacid comprises the isolated nucleic acid of claim
 6. 10. An isolatedcontiguous nucleic acid comprising a first nucleic acid encoding a firsttherapeutic polypeptide and a second nucleic acid encoding a secondtherapeutic polypeptide, wherein the first nucleic acid comprises theisolated nucleic acid of claim
 7. 11. An isolated contiguous nucleicacid comprising a first nucleic acid encoding a first therapeuticpolypeptide and a second nucleic acid encoding a second therapeuticpolypeptide, wherein the first nucleic acid comprises the isolatednucleic acid of claim
 8. 12. The isolated nucleic acid of claim 9,wherein the second therapeutic polypeptide is a cytokine, a growthfactor, or a chemokine.
 13. The isolated nucleic acid of claim 10,wherein the second therapeutic polypeptide is a cytokine, a growthfactor, or a chemokine.
 14. The isolated nucleic acid of claim 11,wherein the second therapeutic polypeptide is a cytokine, a growthfactor, or a chemokine.
 15. The isolated nucleic acid of claim 9,wherein the second therapeutic polypeptide is an IL10 polypeptide. 16.The isolated nucleic acid of claim 10, wherein the second therapeuticpolypeptide is an IL10 polypeptide.
 17. The isolated nucleic acid ofclaim 11, wherein the second therapeutic polypeptide is an IL10polypeptide.
 18. The isolated nucleic acid of claim 6, wherein thenucleic acid sequence encoding the variant FOXP polypeptide isoperatively linked to a promoter.
 19. The isolated nucleic acid of claim18, wherein the promoter is a constitutive promoter.
 20. The isolatednucleic acid of claim 18, wherein the promoter is a tissue-specificpromoter or a pathology-specific promoter.
 21. The isolated nucleic acidof claim 18, wherein the promoter is activated by sheer stress ordyslipidemia.
 22. The isolated nucleic acid of claim 18, wherein thepromoter is activated by Angiotensin-2 (Ang II), Lipopolysaccharides(LPS), Oxidized Low Density Lipoprotein (Ox-LDL), or carbamylated LDL.23. The isolated nucleic acid of claim 9, wherein the first nucleic acidand the second nucleic acid are operatively linked to a promoter. 24.The isolated nucleic acid of claim 9, wherein the first nucleic acid isoperatively linked to a first promoter and the second nucleic acid isoperatively linked to a second promoter.
 25. The isolated nucleic acidof claim 23, wherein the promoter is a constitutive promoter.
 26. Theisolated nucleic acid of claim 23, wherein the promoter is atissue-specific promoter or a pathology-specific promoter.
 27. Theisolated nucleic acid of claim 23, wherein the promoter is activated bysheer stress or dyslipidemia.
 28. The isolated nucleic acid of claim 23,wherein the promoter is activated by Angiotensin-2 (Ang II),Lipopolysaccharides (LPS), Oxidized Low Density Lipoprotein (Ox-LDL), orcarbamylated LDL.
 29. The isolated nucleic acid of claim 18, wherein thepromoter comprises at least one NFκB binding site, at least one Oct1binding site, or at least one NFκB binding site and at least one Oct1binding site.
 30. The isolated nucleic acid of claim 23, wherein thepromoter comprises at least one NFκB binding site, at least one Oct1binding site, or at least one NFκB binding site and at least one Oct1binding site.